Team:TU-Munich/Notebook/Labjournal

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Labjournal

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Week 1 22.4-28.4
Week 2 29.4-05.5
Week 3 06.5-12.5
Week 4 13.5-19.5
Week 5 20.5-26.5
Week 6 27.5-02.6
Week 7 03.6-09.6
Week 8 10.6-16.6
Week 9 17.6-23.6
Week 10 24.6-30.6
Week 11 01.7-07.7
Week 12 08.7-14.7
Week 13 15.7-21.7
Week 14 22.7-28.7
Week 15 29.7-04.8
Week 16 05.8-11.8
Week 17 12.8-18.8
Week 18 19.8-25.8
Week 19 26.8-01.9.
1 kbp GeneRuler:

100 bp GeneRuler:

PageRuler Plus:

Week 1

Monday, April 22nd

Transformation of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Investigator: Jeff, Rosario

Aim of the experiment: Transformation of Phytochrome B for protein fusion.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 2 µl of DNA was added to 100 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Miniprep of pTUM100 with pGAL, pTEF1, pTEF2, pADH and RFC25 compatible RFP generator

Investigator: Jeff, Rosario

Aim of the experiment: Miniprep of pTUM100 with pGAL, pTEF1, pTEF2, pADH and RFC25 compatible RFP generator

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Sequencing of RFP-Generator (RFC25, pSB1C3)

Investigator: Jeff, Rosario

Aim of the experiment: Sequencing of RFP-Generator (RFC25, pSB1C3)

Procedure:

Sequencing batch were prepared after manufacturer's protocol. (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer)

Tuesday, April 23rd

Picking of of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Investigator: Jeff, Rosario, Florian

Aim of the experiment: Picking of of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Procedure:

  • pSB1C3 plasmid with BBa_K801031 (PhyB 2 - 908 aa, RFC25): Colonies were picked from chloramphenicol plates.
  • Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
  • 4 colonies were picked.
  • These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight

Analytical digestion and gelelectrophoresis of RFP-generator (RFC25, pSB1C3, P4 & P5)

Investigator: Jeff, Rosario, Florian

Aim of the experiment: Analytical digestion and gelelectrophoresis of RFP-generator (RFC25, pSB1C3, P4 & P5).


Procedure:

  • Batch for analytical digestion for P4 with NgoMIV+AgeI-HF
volume reagent
2.5 µl Plasmid DNA P4
2 µl NEBuffer 4 (10x)
0.25 µl NgoMIV (10 U/µl)
0.25 µl AgeI-HF (20 U/µl)
15 µl ddH2O
=20 µl TOTAL
  • Batch for analytical digestion for P5 with NgoMIV+AgeI-HF
volume reagent
2.5 µl Plasmid DNA P5
2 µl NEBuffer 4 (10x)
0.25 µl NgoMIV (10 U/µl)
0.25 µl AgeI-HF (20 U/µl)
15 µl ddH2O
=20 µl TOTAL
  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.

Results:

1 kbp ladder DNA ladder P4 P5
Mutation successful Mutation successful!
  • Parts are compliant and do not contain RFC25 forbidden restriction sites.


TUM13 20130423 RFP Generator RFC25 AgeI NgoMIV.png

Sequencing of pTUM vectors with pGAL, pADH, pTEF1, pTEF2

Investigator: Jeff, Rosario, Florian

Aim of the experiment: Sequencing of pTUM vectors with pGAL, pADH, pTEF1, pTEF2

Procedure:

Sequencing batch were prepared after manufacturer's protocol. (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different vectors we sequenced received the following barcodes:

- ADH in pTUM100: FR01002265

- TEF1 in pTUM100: FR01002266

- TEF2 in pTUM100: FR01002266

- GAL in pTUM100: FR01002268


Sequencing of TEF2 in pTUM100 was not interpretable. The other sequences were consistent with the sequences in the parts registry.

Wednesday, April 24th

Miniprep of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Investigator: Jeff, Florian

Aim of the experiment: Miniprep of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3).

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analytical digestion and gelelectrophoresis of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3), P7 - P10

Investigator: Jeff, Florian

Aim of the experiment: Analytical digestion and gelelectrophoresis of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3), P7 - P10.

Procedure:

  • Batch for analytical digestion for P7 with NgoMIV+AgeI-HF
volume reagent
2.5 µl Plasmid DNA P7
2 µl NEBuffer 4 (10x)
0.25 µl NgoMIV (10 U/µl)
0.25 µl AgeI-HF (20 U/µl)
15 µl ddH2O
=20 µl TOTAL
  • Batch for analytical digestion for P8 with NgoMIV+AgeI-HF
volume reagent
2.5 µl Plasmid DNA P8
2 µl NEBuffer 4 (10x)
0.25 µl NgoMIV (10 U/µl)
0.25 µl AgeI-HF (20 U/µl)
15 µl ddH2O
=20 µl TOTAL
  • Batch for analytical digestion for P9 with NgoMIV+AgeI-HF
volume reagent
2.5 µl Plasmid DNA P9
2 µl NEBuffer 4 (10x)
0.25 µl NgoMIV (10 U/µl)
0.25 µl AgeI-HF (20 U/µl)
15 µl ddH2O
=20 µl TOTAL
  • Batch for analytical digestion for P10 with NgoMIV+AgeI-HF
volume reagent
2.5 µl Plasmid DNA P10
2 µl NEBuffer 4 (10x)
0.25 µl NgoMIV (10 U/µl)
0.25 µl AgeI-HF (20 U/µl)
15 µl ddH2O
=20 µl TOTAL
  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.

Results:

1 kbp ladder DNA ladder P7 P8 P9 P10
Part is correct Part is correct Part is correct Part is correct


TUM13 20130424 PhytochromeB RFC25 AgeI NgoMIV.png

Transformation of E. coli XL1 blue with EreA and EreB (pDEST14)

Investigator: Jeff, Florian

Aim of the experiment: Transformation of E. coli XL1 blue with EreA and EreB (pDEST14).

Procedure:

  • Plasmid DNA was received dried in paper from McMaster University.
  • DNA was resuspended in ddH2O
  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 2 µl of DNA was added to 100 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The transformated cells were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on chlorampenicol and ampicillin plates.

Week 2

Monday, April 29th

Miniprep of pRK792, pRK793, pRIL, ereA, ereB

Investigator: Rosario

Aim of the experiment: Miniprep of pRK792, pRK793, pRIL, ereA, ereB

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
  • Concentrations were determined by measuring the absorption:
Plasmid c [ng/µl]
pRK792 214,5
pRK793 154,3
pRIL 6,1
ereA 183,4
ereB 98,3
  • The procedure will have to be repeated for pRIL

Midiprep of RFC25 compatible RFP generator in (pSB1C3)

Investigator: Andreas, Florian

Aim of the experiment: Midiprep of RFC25 compatible RFP generator in (pSB1C3)

Procedure:

  • Midiprep was performed after manufacturer's protocol (QIAprep Midiprep, QIAGEN)

Tuesday, April 30th

Preparative digestion and gelelectrophoresis of mINF1 and Leptin with HindIII and EheI

Investigator: Louise, Johanna

Aim of the experiment: Testing the enzymes, since the earlier digestion with probably older enzymes did not work (no bands on gel) .

Procedure:

  • Batch for preparative digestion for mINF1 with HindIII and EheI
volume reagent
10 µl Plasmid DNA mINF11
5 µl Tango Buffer (10x)
2 µl HindIII (10 U/µl)
3 µl EheI (10 U/µl)
30 µl ddH2O
=50 µl TOTAL
  • Batch for preparative digestion for Leptin with HindIII and EheI
volume reagent
20 µl Plasmid DNA Leptin
5 µl Tango Buffer (10x)
2 µl HindIII (10 U/µl)
3 µl EheI (10 U/µl)
20 µl ddH2O
=50 µl TOTAL
  • Incubation for 3 h at 37 °C.

Transformation of E. coli XL1 blue with PSH21 (P17)

Investigator: Johanna, Louise

Aim of the experiment: Transformation of E. coli XL1 blue with PSH21.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice for 10 minutes.
  • 5 µl of DNA was added to 250 µl of competent cells and gently mixed.
  • 60 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding the DNA to 2 ml LB-medium.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The transformated cells were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on chlorampenicol and ampicillin plates.

Thursday, May 2nd

Miniprep of pSH21 (pAutoRex8) containing AlcR/AlcA promotor system

Investigator: Katrin

Aim of the experiment: Miniprep of pSH21 (pAutoRex8) containing AlcR/AlcA promotor system

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
  • The concentration was measured (900 ng/µl)


Friday, May 3rd

Transformation of E. coli XL1 blue with pSB1C3-GFP, pSB1C3-mkate2, pSB1C3-mCherry

Investigator: Andreas

Aim of the experiment: Transformation of E. coli XL1 blue with pSB1C3-GFP, pSB1C3-mkate2, pSB1C3-mCherry.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice for 10 minutes.
  • 5 µl of DNA was added to 250 µl of competent cells and gently mixed.
  • 60 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding the DNA to 2 ml LB-medium.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The transformated cells were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on chlorampenicol and ampicillin plates.

Plating of received E. coli containing biobricks

Investigator: Jeff, Andreas

Aim of the experiment: The received biobricks were already transformed in E. coli and were in an agar stabs. These E. coli cells were transferred with an inoculation loop on antibiotic selection plates and were incubated over night.

Operational sequence:

  • Bacterias containing plasmids with biobricks were transferred with a sterile inoculation loop on antibiotic plates and were incubated at 37 °C overnight.
  • The biobricks were:
Name Function
[[http://partsregistry.org/Part:BBa_K157004 BBa_K157004]] Fluoresceine A -binding derivative of a Lipocalin
[[http://partsregistry.org/Part:BBa_K863000 BBa_K863000]] bpul (laccase from Bacillus pumilus) with T7 promoter, RBS and HIS tag
[[http://partsregistry.org/Part:BBa_K909009 BBa_K909009]] cDNA of UV-B sensing protein UVR8 from Arabidopsis thaliana
[[http://partsregistry.org/Part:BBa_K337013 BBa_K337013]] constitutive promoter SV40
[[http://partsregistry.org/Part:BBa_K747096 BBa_K747096]] constitutive promoter CMV
[[http://partsregistry.org/Part:BBa_K414002 BBa_K414002]] constitutive promoter CaMV35S
[[http://partsregistry.org/Part:BBa_K147002 BBa_K147002]] xylE gene coding for catechol-1,2-dioxygenase
[[http://partsregistry.org/Part:BBa_E2020 BBa_E2020]] Cerulean
[[http://partsregistry.org/Part:BBa_K404319 BBa_K404319]] mCFP
[[http://partsregistry.org/Part:BBa_E0040 BBa_E0040]] GFPmut3b
[[http://partsregistry.org/Part:BBa_K592010 BBa_K592010]] amilGFP
[[http://partsregistry.org/Part:BBa_E2050 BBa_E2050]] mOrange
[[http://partsregistry.org/Part:BBa_K399001 BBa_K399001]] mStrawberry
[[http://partsregistry.org/Part:BBa_E1010 BBa_E1010]] mRFP1
[[http://partsregistry.org/Part:BBa_E2060 BBa_E2060]] mCherry
[[http://partsregistry.org/Part:BBa_K592012 BBa_K592012]] eforRed
[[http://partsregistry.org/Part:BBa_K592011 BBa_K592011]] cjBlue
[[http://partsregistry.org/Part:BBa_K864401 BBa_K864401]] aeBlue
[[http://partsregistry.org/Part:BBa_K592009 BBa_K592009]] amilCP, blue chromoprotein
[[http://partsregistry.org/wiki/index.php?title=Part:BBa_K414000 BBa_K414000]] RD29A Promoter + Strong Plant Kozak (RBS) + RFP
[[http://partsregistry.org/wiki/index.php?title=Part:BBa_K414006 BBa_K414006]] 35S Promoter + (Strong Plant RBS + GFP) From K414001
[[http://partsregistry.org/wiki/index.php?title=Part:BBa_K414007 BBa_K414007]] DREB1C promoter
[[http://partsregistry.org/wiki/index.php?title=Part:BBa_K678001 BBa_K678001]] alcA promoter
[[http://partsregistry.org/Part:BBa_K243004 BBa_K243004]] Short linker
[[http://partsregistry.org/Part:BBa_K243005 BBa_K243005]] Middle linker
[[http://partsregistry.org/Part:BBa_K243006 BBa_K243006]] Long Linker
[[http://partsregistry.org/Part:BBa_K243029 BBa_K243029]] GSAT Linker
[[http://partsregistry.org/Part:BBa_K243030 BBa_K243030]] SEG Linker

Sunday, May 5th

Picking of the plated E. coli containing biobricks and the parts received from LMU

Investigator: Florian

Aim of the experiment: The colonies from the plates were transferred into liquid LB medium, so that minipreps can be performed the next day.

Operational sequence:

  • Tubes with 5 ml LB medium and the corresponding antibiotic were prepared.
  • A single colony from each plate was transferred into a tube with a pipet tip.
  • The biobricks were:
Label Name Function
p19 mCherry in pSB1C3
p20 [[http://partsregistry.org/Part:BBa_K823039 BBa_K823039]] GFP in pSB1C3
p21 [[http://partsregistry.org/Part:BBa_K823029 BBa_K823029]] mKate2 in pSB1C3
p22 [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K414006 BBa_K414006]] 35S Promoter + (Strong Plant RBS + GFP) From K414001
p23 [[http://partsregistry.org/Part:BBa_K414002 BBa_K414002]] constitutive promoter CaMV35S
p24 [[http://partsregistry.org/Part:BBa_K909009 BBa_K909009]] cDNA of UV-B sensing protein UVR8 from Arabidopsis thaliana
p25 [[http://partsregistry.org/Part:BBa_K399001 BBa_K399001]] mStrawberry
p26 [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K414007 BBa_K414007]] DREB1C promoter
p27 [[http://partsregistry.org/Part:BBa_K863000 BBa_K863000]] bpul (laccase from Bacillus pumilus) with T7 promoter, RBS and HIS tag
p28 [[http://partsregistry.org/Part:BBa_K337013 BBa_K337013]] constitutive promoter SV40
p29 [[http://partsregistry.org/Part:BBa_K592011 BBa_K592011]] cjBlue
p30 [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K678001 BBa_K678001]] alcA promoter
p31 [[http://partsregistry.org/Part:BBa_K592012 BBa_K592012]] eforRed
p32 [[http://partsregistry.org/Part:BBa_K592010 BBa_K592010]] amilGFP
p33 [[http://partsregistry.org/Part:BBa_K864401 BBa_K864401]] aeBlue
p34 [[http://partsregistry.org/Part:BBa_K404319 BBa_K404319]] mCFP
p35 [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K414000 BBa_K414000]] RD29A Promoter + Strong Plant Kozak (RBS) + RFP
p36 [[http://partsregistry.org/Part:BBa_K592009 BBa_K592009]] amilCP, blue chromoprotein
p37 [[http://partsregistry.org/Part:BBa_K747096 BBa_K747096]] constitutive promoter CMV
p38 [[http://partsregistry.org/Part:BBa_K147002 BBa_K147002]] XylE dioxygenase (catechol dioxygenase)
p39 [[http://partsregistry.org/Part:BBa_K243005 BBa_K243005]] Middle linker
p40 [[http://partsregistry.org/Part:BBa_K243029 BBa_K243029]] GSAT Linker
p41 [[http://partsregistry.org/Part:BBa_E0040 BBa_E0040]] GFPmut3b
p42 [[http://partsregistry.org/Part:BBa_K243006 BBa_K243006]] Long Linker
p43 [[http://partsregistry.org/Part:BBa_K243030 BBa_K243030]] SEG Linker
p44 [[http://partsregistry.org/Part:BBa_K243004 BBa_K243004]] Short linker
p45 [[http://partsregistry.org/Part:BBa_K157004 BBa_K157004]] Fluoresceine A -binding derivative of a Lipocalin
p46 [[http://partsregistry.org/Part:BBa_E2060 BBa_E2060]] mCherry
p47 [[http://partsregistry.org/Part:BBa_E2020 BBa_E2020]] Cerulean
p48 [[http://partsregistry.org/Part:BBa_E1010 BBa_E1010]] mRFP1
p49 [[http://partsregistry.org/Part:BBa_E2050 BBa_E2050]] mOrange
  • There were no colonies on the plate with K864401 (p33), therefore the LB medium could not be infected
  • The tubes were placed into the incubator at 37 °C

Week 3

Monday, May 6th

Miniprep of the prepared E. coli containing biobricks and the parts from LMU

Investigators: Florian, Johanna, Jefferey

Aim of the experiment: A miniprep of the overnight culture of our E. coli containig our biobricks and LMU parts was performed using the Qiagen miniprep kit.

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
  • Resulting concentrations were determined by measuring the absorption:
Label Name Concentration [ng/µl]
p19 mCherry in pSB1C3 40,2
p20 GFP in pSB1C3 58,5
p21 mKate2 in pSB1C3 131,3
p22 35S Promoter + (Strong Plant RBS + GFP) From K414001 42,8
p23 constitutive promoter CaMV35S 39,3
p24 cDNA of UV-B sensing protein UVR8 from Arabidopsis thaliana 30,0
p25 mStrawberry 46,6
p26 DREB1C promoter 34,8
p27 bpul (laccase from Bacillus pumilus) with T7 promoter, RBS and HIS tag 52,5
p28 constitutive promoter SV40 58,7
p29 cjBlue 44,4
p30 alcA promoter 26,9
p31 eforRed 64,8
p32 amilGFP 59,3
p34 mCFP 78,4
p35 RD29A Promoter + Strong Plant Kozak (RBS) + RFP 61,2
p36 amilCP, blue chromoprotein 89,2
p37 constitutive promoter CMV 83,6
p38 XylE dioxygenase (catechol dioxygenase) 57,4
p39 Middle linker 64,3
p40 GSAT Linker 109,6
p41 GFPmut3b 118,1
p42 Long Linker 133,0
p43 SEG Linker 107,5
p44 Short linker 60,2
p45 Fluoresceine A -binding derivative of a Lipocalin 82,7
p46 mCherry 230,2
p47 Cerulean 25,7
p48 mRFP1 299,3
p49 mOrange 181,2


Sequencing of FluA, SV40, CaMV35S, xylE, LMU mKate2, LMU GFP, LMU mCherry, DREB1C, RD29A

Investigators: Andreas, Florian, Johanna, Jefferey, Rosario

Aim of the experiment: Sequencing of genes for which no prior sequencing results were available.

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:


Label Name Barcode
p45 FluA FR01002355
p28 SV40 FR01002356
p23 CaMV35S FR01002357
p38 xylE FR01002358
p21 LMU mKate2 FR01002359
p20 LMU GFP FR01002360
p19 LMU mCherry FR01002345
p46 mCherry FR01002346
p26 DREB1C FR01002347
p35 RD29A FR01002348

Preparation of ordered primers

Investigators: Andreas, Jefferey, Rosario

Aim of the experiment: Dissolving of primer pallets to provide a concentration of 100 pmol/µl

Procedure: We added an amount of water which gave us a final primer concentration of 100 pmol/µl.

Primers were labeled as follows:

Label Oligonr.
6 EreA_for
7 EreA_rev
8 EreA_SDM1_for
9 EreA_SDM1_rev
10 EreA_SDM2_for
11 EreA_SDM2_rev
12 EreA_SDM3_for
13 EreA_SDM3_rev
14 EreB_for
15 EreB_rev
16 EreB_SDM1_for
17 EreB_SDM1_rev
18 EreB_SDM2_for
19 EreB_SDM2_rev
20 EreB_SDM3_for
21 EreB_SDM3_rev

Tuesday, May 7th

PCR, purification and analytical geleletrophoresis of EreA (P15) and EreB (P16)

Investigator: Jeff, Louise, Florian, Rosario, Andi, Johanna

Aim of the experiment: PCR of EreA (P15) and EreB (P16).

Procedure:

Operational sequence:

  • PCR reaction mixture
volume reagent
10 µl 5x OneTaq Standard Reaction Buffer
1 µl 10 mM dNTPs
1 µl 10 µM Forward Primer (P15: O46 (EreA_for); P16: O31 (EreB_for))
1 µl 10 µM Reverse Primer (P15: O47 (EreA_rev); P16: O32 (EreB_rev))
0.25 µL OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl Plasmid DNA (P15; P16)
35.75 µL ddH2O Water
=50 µL TOTAL
  • Mix with pipette
  • The gradient PCR program was performed after following scheme with following conditions (Tm=54 °C; ΔG=2 °C; P15 in row 11(=52 °C); P16 in row 1 (=54.0 °C)):
Initial denaturation 94 °C 30 s
30 cycles 94 °C 30 s
Tm=54 °C; ΔG=2 °C 60 s
68 °C 80 s
Final extension 68 °C 5 min
Hold 4 °C infinite
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
  • 9 µl of the purified PCR products were mixed with 1 µl DNA loading buffer (10x) for analytical gelelectrophoresis
  • Analytical gelelectrophoresis was performed at 90 V for 60 min in 1% agarose gel.
1 kbp ladder F1 F2
Fragment-size like expected Fragment-size like expected

TUM13 20130507 P15 P16 PCR.png

Preparative digestion and gelelectrophoresis of PhyB (P9) and PIF3-20aa linker (P50) with AgeI and PstI as well as 20aalinker-PIF3 (P51) with EcoRI and NgoMIV

Investigator: Jeff, Andi, Florian, Rosario, Louise, Johanna

Aim of the experiment: Preparative digestion and gelelectrophoresis of PhyB (P9) and PIF3-20aa linker (P50) with AgeI and PstI as well as 20aalinker-PIF3 (P51) with EcoRI and NgoMIV .

Procedure:

  • Batch for preparative digestion of PhyB (P9) and PIF3-20aa linker (P50) with AgeI and PstI
volume reagent
20 µl Plasmid DNA P9/P50
4 µl NEBuffer 4
0.4 µl BSA (100x)
1 µl AgeI-HF
1 µl PstI-HF
13.6 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of 20aalinker-PIF3 (P51) with EcoRI and NgoMIV
volume reagent
20 µl Plasmid DNA P51
4 µl NEBuffer 4
1 µl EcoRI-HF
1 µl NgoMIV
14 µl ddH2O
=40 µl TOTAL
  • Incubation for 3 h at 37 °C.
  • 4 µl of DNA loading buffer (10x) were added to the reaction batches after digestion and were loaded on an 1% low-melting agarose gel for preparative gelelectrophoresis.
  • Preparative gelelectrophoresis was performed at 70 V for 90 min.
P9 digestion = F3 1 kbp ladder P50 digestion = F4 P51 digestion = F5
Fragment-size like expected; band was cut out Fragment-size like expected; band was cut out Fragment-size like expected; band was cut out

TUM13 20130507 P9 AgeI.PstI P50 AgeI.PstI P51 EcoRI.NgoMIV.png

  • Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.

Wednesday, May 8th

Preparative digestion and gelelectrophoresis of PCR product of EreA (F1) and EreB (F2) with XbaI & AgeI

Investigator: Jeff, Andi, Florian, Rosario, Louise

Aim of the experiment: Preparative digestion and gelelectrophoresis of PCR product of EreA (F1) and EreB (F2) with XbaI & AgeI.

Procedure:

  • Batch for preparative digestion of PCR product of EreA (F1) and EreB (F2) with XbaI & AgeI.
volume reagent
25 µl PCR product F1/F2
5 µl NEBuffer 4
0.5 µl BSA (100x)
1 µl AgeI-HF
1 µl XbaI
17.5 µl ddH2O
=50 µl TOTAL
  • Batch for preparative digestion of P52 for preperation of pSB1C3 vector:
volume reagent
20 µl Plasmid DNA P52
4 µl NEBuffer 4
0.4 µl BSA (100x)
1 µl AgeI-HF
1 µl XbaI
13.6 µl ddH2O
=40 µl TOTAL
  • Incubation for 3 h at 37 °C.
  • 5 µl of DNA loading buffer (10x) were added to the reaction batches for F1 & F2 and 4 µl of DNA loading buffer (10x) were added to the reaction batches for Pxx after digestion and were loaded on an 1% agarose gel for preparative gelelectrophoresis.
  • Preparative gelelectrophoresis was performed at 90 V for 60 min.
F1 digestion = F6 F2 digestion = F7 1 kbp ladder P52 digestion = F8
Length was like expected Length was like expected Length was like expected; the lower band was extracted

TUM13 20130508 F1 F2 P52 Xb.png

  • Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.

Ligation of F8+F6 (EreA in pSB1C3), F8+F7 (EreB in pSB1C3)

Investigator: Jeff, Andi, Florian, Rosario, Louise

Aim of the experiment: Ligation of F8+F6 (EreA in pSB1C3), F8+F7 (EreB in pSB1C3). Quickchanges have still to be performed.

Procedure:

Ligation batch for F8+F6 (EreA in pSB1C3), F8+F7 (EreB in pSB1C3):

  • Ligation batch for F8+F6
volume reagent
1.35 µl F8 (74.3 ng/µl, 2086 bp)
6.76 µl F6 (25.9 ng/µl, 1218 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
8.89 µl ddH2O
=20 µl TOTAL
  • Ligation batch for F8+F7
volume reagent
1.35 µl F8 (74.3 ng/µl, 2086 bp)
8.78 µl F7 (20.6 ng/µl, 1257 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
6.87 µl ddH2O
=20 µl TOTAL
  • Negative control was als prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batches.
  • Cycled ligation has been performed after following protocol in a thermocycler:
100 cycles 12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
Hold 16 °C infinite


Lid temperature = 37 °C

Midiprep of cloning vector RFC25 RFP generating device

Investigators: Andreas, Rosario

Aim of the experiment: Midiprep of our cloning vector RFC25 RFP generating device.

Procedure: Midiprep was performed after manufacturer's protocol (QIAprep Midiprep, QIAGEN)

The yield of our vector was 1854 ng.

Thursday, May 9th

Transformation of E. coli XL1 blue with F8(pSB1C3)+F6(EreA) and F8(pSB1C3)+F7(EreB)

Investigator: Florian, Jeff, Louise, Katrin, Ingmar

Aim of the experiment: Transformation of E. coli XL1 blue with F8(pSB1C3)+F6(EreA) and F8(pSB1C3)+F7(EreB). Quickchanges has now to be performed to remove forbidden restriction sites.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of ligation products F8+F6, F8+F7, F8 negative control were added to 100 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Friday, May 10th

Quick Change mutagenesis to remove mutations found by sequencing in our gene constructs

Investigator: Andi, Jeff, Rosario

Aim of the experiment: Deletion of found mutations.

Operational sequence

1. PCR general setup

Reaction batch 1

volume reagent
2.5 µl 10x Pfu Ultra II buffer
4 µl DNA template
1 µl 1:10 dilution of appropriate Oligo (= 5 pmol)
16.5 µl ddH2O
0.5 µl dNTP mix
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)

Reaction batch 2

volume reagent
2.5 µl 10x Pfu Ultra II buffer
4 µl DNA template
1 µl 1:10 dilution of appropriate Oligo (= 5 pmol)
16.5 µl ddH2O
0.5 µl dNTP mix
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)


PCR cycling parameters

Segment Cycles Temperature Time
1 1 95 °C 30 sec
2 12 95 °C 30 s
55 °C 1 min
68 °C 6 min
  • Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied.
  • After the PCR was finished 1 µl of the DpnI restriction enzyme (10 U/µl)was added
  • Now the reaction was mixed gently and thoroughly, spinned down in a microcentrifuge for 1 minute, and immediately incubated at 37 °C for 1 hour to digest the parental supercoiled dsDNA


2. Used constructs and primers

Quickchange number Plasmid number Geneconstruct Oligo number
QC 1P27BPul_Laccase+ pSB1C3O26 & O27

Picking of transformed Plasmids F8 + F6

Investigator: Andi

Aim of the study: Picking from the overnight transformation of the ligated F8+F6, to see whether ligation is successful or not.

Operational sequence:

  • Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)

PCR of TEV (P14, pRK793)

Investigator: Katrin, Jeff

Aim of the experiment: PCR, purification and analytical geleletrophoresis of TEV (P14, pRK793) to produce TEV with RFC25 pre- and suffx (still contains forbidden SpeI-site).

Procedure:

Operational sequence:

  • PCR reaction mixture
volume reagent
10 µl 5x OneTaq Standard Reaction Buffer
1 µl 10 mM dNTPs
1 µl 10 µM Forward Primer (O34 (TEV_fw))
1 µl 10 µM Reverse Primer (O35 (TEV_rv))
0.25 µL OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl Plasmid DNA (P14)
35.75 µL ddH2O Water
=50 µL TOTAL
  • Mix with pipette
  • The PCR program was performed after following scheme with following conditions:
Initial denaturation 94 °C 30 s
30 cycles 94 °C 30 s
55 °C 60 s
68 °C 150 s
Final extension 68 °C 5 min
Hold 4 °C infinite
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
  • Resulting product was labeled as F12.

Digestion of P61 with S1 Nuclease

Investigator: Katrin

Oligohybridization of O22 (MinMCS_for) and O23 (MinMCS_rev)

Investigator: Andi

Aim of the experiment: Oligohybridization of MinMCS_for (O22) and MinMCS_rev (O23

Procedure:

  • 25 µL of 100 pmol/µl of MiniMCS fw and 25 µL of 100 pmol/µl MiniMCS rev
  • Heating up to 95 °C for 5 min
  • Cooling at RT in a styropor box overnight

Preparation of ordered primers

Investigators: Andreas, Rosario, Katrin, Ingmar

Aim of the experiment: Dissolving of primer pallets to provide a concentration of 100 pmol/µl

Procedure: We added an amount of water which gave us a final primer concentration of 100 pmol/µl.

Primers were labeled as follows:

Label Oligonr.
24 BPU_for
25 BPU_rev
26 BPU_SDM1_for
27 BPU_SDM1_rev
28 BPU_SDM2_for
29 BPU_SDM2_rev
30 BPU_SDM3_for
31 BPU_SDM3_rev
32 BPU_SDM4_for
33 BPU_SDM4_rev
34 TEV_for
35 TEV_rev
36 TEV_t387c_for
37 TEV_t387c_rev
38 N_Split_TEV_for
39 N_Split_TEV_rev
40 C_Split_TEV_for
41 C_Split_TEV_rev
42 PIF6_2-100_for
43 PIF6_2-100_rev
44 p104AgeI a71g fw
45 p104AgeI a71g rev
46 xylE_for
47 xylE_rev
48 xylE_c312a_for
49 xylE_c312a_rev
50 xylE_g483a_for
51 xylE_g483a_rev
52 xylE_a834g_for
53 xylE_a834g_rev

Saturday, May 11th

Preparative digestion of PCR product of S219V pRK793 (F12) with XbaI & AgeI

Investigator: Louise

Aim of the experiment: Preparative digestion of PCR product of S219V pRK793 (F12) with XbaI & AgeI. Quickchange has still to be performed (forbidden SpeI restriction site).

Procedure:

  • Batch for preparative digestion of PCR product S219V pRK793 (F12) with XbaI & AgeI.
volume reagent
20 µl PCR product F12
4 µl NEBuffer 4
0.4 µl BSA (100x)
1 µl AgeI-HF
1 µl XbaI
13.6 µl ddH2O
=40 µl TOTAL
  • Incubation for 3 h at 37 °C
  • Digested products were purified to remove nucleotides and enzymes with QIAquick PCR Purification Kit, QIAGEN.
  • The digested fragment was labelled F15

Miniprep of overnight culture of transformated E.~coli XL1-Blue with F8 (pSB1C3) + F6 (EreA)

Investigator: Louise

Aim of the experiment: Miniprep of overnight culture of transformated E.~coli XL1-Blue with F8 (pSB1C3) + F6 (EreA) of 2 clones picked the day before

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
  • The plasmids were labelled as P63 and P64

Analytical digestion and gelelectrophoresis of P63 and P64 with AgeI-HF and XbaI

Investigator: Louise

Aim of the experiment: Analytical digestion and gelelectrophoresis of P63 and P64 with AgeI-HF and XbaI

Procedure:

  • Batch for analytical digestion of P63 and P64 with AgeI-HF and XbaI
volume reagent
2.5 µl Plasmid DNA P63/P64
2 µl NEBuffer 4 (10x)
0.2 µl BSA (100x)
0.25 µl XbaI(10 U/µl)
0.25 µl AgeI-HF (20 U/µl)
14.8 µl ddH2O
=20 µl TOTAL
  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.


Analytical digestion of P63 with AgeI-HF and XbaI 1 kbp ladder Analytical digestion of P64 with AgeI-HF and XbaI
Insertion successful Insertion successful

TUM13 20130511 P63 P64 XbaI.png

Preparative digestion of PCR product of EreB (F2) with XbaI & AgeI

Investigator: Louise, Johanna

Aim of the experiment: Preparative digestion of PCR product EreB (F2) with XbaI & AgeI (second try since there did not grow any transformed bacteria in the first time).

Procedure:

  • Batch for preparative digestion of PCR product and EreB (F2) with XbaI & AgeI.
volume reagent
20 µl PCR product F2
5 µl NEBuffer 4
0.5 µl BSA (100x)
1 µl AgeI-HF
1 µl XbaI
22.5 µl ddH2O
=50 µl TOTAL
  • Incubation for 3 h at 37 °C
  • The digested fragment was labelled F14

Purification and ligation of digested PCR product EreB (F14)

Investigator: Louise, Rosario

Aim of the experiment: PCR Purification and ligation of digested PCR product EreB (F14).

Procedure: PCR Purification of EreB PCR (F14)

  • To purify the digested Fragment(F14) the QIAquick PCR Purification Kit was used. The concentration of the purified product was found to be 5 ng/µl via Nano drop.

Ligation of F14 + F8(pSB1C3)

  • Ligation batch for F8+F14 (positive control)
volume reagent
1.35 µl F8 (74.3 ng/µl, 2086 bp)
15.65 µl F14 (5 ng/µl, 1257 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
  • Cycled ligation has been performed after following protocol in a thermocycler:
99 cycles 12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
Hold 16 °C infinite


Lid temperature = 37 °C

Transformation of ligation products of F10 + F11 into E.coli Xl1-Blue

Investigator: Andi

Aim of the experiment:Transformation of the ligation products of F10 + F11 and the negative control in pTUM104 in XL1 Blue.

Operation Sequence

  • melting of 2x 200 µl Ca-competent E.coli XL1-Blue cells on ice
  • Preparing 4 Tubes with 100 µl of Ca-competent E.coli XL1-Blue cells
  • addition of 5 µl of the ligation products
  • incubation for 30 min on ice
  • heat shock for 5 min at 37 °C
  • transfer of cells to 1 ml LB-medium without antibiotics and incubate at 37°C and 180 rpm for 30 min
  • plate 100 µl on an Amp-LB-plate
  • sediment the leftover in a centrifuge (30 - 60 sec, 13 000 rpm) and resuspend the sediment in 100 µl LB-medium and plate it as well on an Amp-LB-plate

Transformation of Quickchange Product P62 into E.coli Xl1-Blue

Investigator: Andi

Aim of the experiment:Transformation of the Quickchange Product P62 and the negative control in XL1 Blue.

Operation Sequence

  • melting of 1x 200 µl Ca-competent E.coli XL1-Blue cells on ice
  • Preparing 4 Tubes with 50 µl of Ca-competent E.coli XL1-Blue cells
  • addition of 1 µl of the Quickchange Product
  • incubation for 30 min on ice
  • heat shock for 5 min at 37 °C
  • transfer of cells to 1 ml LB-medium without antibiotics and incubate at 37°C and 180 rpm for 30 min
  • plate 100 µl on an Amp-LB-plate
  • sediment the leftover in a centrifuge (30 - 60 sec, 13 000 rpm) and resuspend the sediment in 100 µl LB-medium and plate it as well on an Amp-LB-plate

Quickchange mutagenesis of pTUM104

Investigator: Ingmar

Aim of the experiment: Sequencing results showed a forbiden AgeI restriction site in this vector. The sdm will delete this restricition site

Procedure: PCR

Reaction batch 1

volume reagent
2.5 µl 10x Pfu Ultra II buffer
4 µl Plasmid P6 template
0.5 µl 1:10 dilution of O44 (10 pmol/µL)
16.5 µl ddH2O
0.5 µl dNTP mix
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)

Reaction batch 2

volume reagent
2.5 µl 10x Pfu Ultra II buffer
4 µl Plasmid P6 template
0.5 µl 1:10 dilution of O45 (10 pmol/µL)
16.5 µl ddH2O
0.5 µl dNTP mix
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment Cycles Temperature Time
1 1 95 °C 30 sec
2 10 95°C 30 sec
55°C 1 min
67°C 6 min
  • Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied.
  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C. The resulting product was named fragmet 14.

Transformation into E.coli Xl1-Blue Operation Sequence

  • melting of 100 µl Ca-competent E.coli XL1-Blue cells on ice
  • addition of 1 µl of the PCR product
  • incubation for 30 min on ice
  • heat shock for 5 min at 37 °C
  • transfer of cells to 1 ml LB-medium without antibiotics and incubate at 37°C and 180 rpm for 30 min
  • plate 100 µl on an Amp-LB-plate
  • sediment the leftover in a centrifuge (30 - 60 sec, 13 000 rpm) and resuspend the sediment in 100 µl LB-medium and plate it as well on an Amp-LB-plate

Sunday, May 12th

Analytical gelelectrophoresis of F15 (digested TEV PCR product)

Investigator: Jeff, Rosario

Aim of the experiment: Analytical gelelectrophoresis of F15 (digested TEV PCR product) to check whether PCR has worked.

Procedure:

  • 5 µl of F15 was mixed with 0.55 µl of DNA loading buffer (10x) and analytical gelelectrophoresis was performed at 1% agarose gel for 60 min at 90 V.

TUM13 20130512 PCR F12.png

  • PCR product has expected length

Transformation of E. coli XL1 blue with ligation product F8+F15 (TEV in pSB1C3, RFC25)

Investigator: Jeff, Rosario

Aim of the experiment: Transformation of E. coli XL1 blue with ligation product F8+F15 (TEV in pSB1C3, RFC25). Further quickchanges still have to be done.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of the ligation products were added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Transformation of E. coli XL1 blue with ligation products F8+F14 (EreB in pSB1C3, RFC25)

Investigator: Jeff, Rosario

Aim of the experiment: Transformation of E. coli XL1 blue with ligation product F8+F14 (EreB in pSB1C3, RFC25). Further quickchanges still have to be done.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of the ligation products were added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Transformation of E. coli XL1 blue with P23

Investigator: Jeff, Rosario

Aim of the experiment: Transformation of E. coli XL1 blue with P23 for insertion of miniMCS.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 2 µl of DNA was added to 100 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Picking of transformed Plasmid F10+F11 (IRES from polio virus, blunt ligated with pSB1C3 for sequencing)

Investigator: Andi

Aim of the study: Picking of transformed Plasmid F10+F11 (AlcR blunt ligated with pSB1C3 for sequencing).

Operational sequence:

  • Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)
  • 10 colonies were picked.

Week 4

Monday, May 13th

Miniprep of overnight culture of transformated E.~coli XL1-Blue with F10+F11

Investigator: Rosario, Jeffery, Johanna, Flo

Aim of the experiment: Miniprep of overnight culture of transformated E.~coli XL1-Blue F10+F11 of 10 clones picked the day before

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analyzing sequencing results

Investigator: Johanna

Aim of the experiment: Comparison of the sequencing results with the Biobrick (BB) sequences.

Procedure: Using Geneious to align the sequencing results with the BB sequences.

LMU GFP in RFC 25(p20)

  • The provided sequence in the parts registry is totally wrong.
  • The comparison of the amino acid seqeunces reveals several point mutations.

TUM13 AlignmentforLMUGFP.png

FluA in RFC 25 (p45)

  • Did not yield sequencing results (does the primer bind ?)
  • No presence of RCF 25 standard restriction enzymes

mCherry in RFC 25 (p46)

  • Did not yield sequencing results (does the primer bind ?)
  • No presence of RCF 25 standard restriction enzymes

CaMV35S in RFC 25 (p23)

  • high identity
  • high match with HQ699544 (Blast)

LMU mKate2in RFC 25 (p21)

  • no continous ORF
  • Did not yield sequencing results

DREB1C promoter in RFC 25 (p26)

  • Present of fluorecent protein gene

xylE in RCF 25 (p38)

  • Did not yield sequencing results

SV40 in RCF 25 (p28)

  • sequencing result agrees with the laccases
  • the sequencing result can't be the one of SV40

Quickchange mutagenesis SDMI of p27

Investigator: Rosario, Jeff, Flo, Andi

Aim of the experiment: Sequencing results showed a forbidden restriction site in this vector. The sdm will delete this restricition site. The resulting Plasmid was labeled 65.

Procedure: PCR

Reaction batch 1

volume reagent
2.5 µl 10x Pfu Ultra II buffer
0.5 µl Plasmid P27 template
0.5 µl 1:10 dilution of O26 (10 pmol/µL)
20. 5 µl ddH2O
0.5 µl dNTP mix
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)

Reaction batch 2

volume reagent
2.5 µl 10x Pfu Ultra II buffer
0.5 µl Plasmid P27 template
0.5 µl 1:10 dilution of O27 (10 pmol/µL)
20.5 µl ddH2O
0.5 µl dNTP mix
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment Cycles Temperature Time
1 1 95 °C 30 sec
2 10 95°C 30 sec
55°C 1 min
67°C 4 min
  • Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied, but the amount of cycles was increased to 20
  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

Quickchange mutagenesis SDMI of p38

Investigator: Jeff, Flo, Andi, Rosario

Aim of the experiment: Sequencing results showed a forbidden restriction site in this vector. The sdm will delete this restricition site. The resulting Plasmid was labeled 67.

Procedure: PCR

Reaction batch 1

volume reagent
2.5 µl 10x Pfu Ultra II buffer
0,5 µl Plasmid P38 template
0.5 µl 1:10 dilution of O48 (10 pmol/µL)
20.5 µl ddH2O
0.5 µl dNTP mix
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)

Reaction batch 2

volume reagent
2.5 µl 10x Pfu Ultra II buffer
0.5 µl Plasmid P38 template
0.5 µl 1:10 dilution of O49 (10 pmol/µL)
20.5 µl ddH2O
0.5 µl dNTP mix
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment Cycles Temperature Time
1 1 95 °C 30 sec
2 10 95°C 30 sec
55°C 1 min
67°C 4 min
  • Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied, but the amount of cycles have been increased to 20
  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

Quickchange mutagenesis SDMI of P63

Investigator: Jeff, Rosario, Flo, Andi

Aim of the experiment: Sequencing results showed a forbidden restriction site in this vector. The sdm will delete this restricition site. The resulting Plasmid was labeled P66.

Procedure: PCR

Reaction batch 1

volume reagent
2.5 µl 10x Pfu Ultra II buffer
0.25 µl Plasmid P63 template; 1:2 dilution
0.5 µl 1:10 dilution of O8 (10 pmol/µL)
20.75 µl ddH2O
0.5 µl dNTP mix
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)

Reaction batch 2

volume reagent
2.5 µl 10x Pfu Ultra II buffer
0.25 µl Plasmid P63 template; 1:2 dilution
0.5 µl 1:10 dilution of O9 (10 pmol/µL)
20.75 µl ddH2O
0.5 µl dNTP mix
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment Cycles Temperature Time
1 1 95 °C 30 sec
2 10 95°C 30 sec
55°C 1 min
67°C 6 min
  • Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied, the amount of cycles was raised to 20
  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

Transformation of E. coli XL1 blue with P65

Investigator: Jeff, Rosario, Johanna, Andi, Flo

Aim of the experiment: Transformation of E. coli XL1 blue with P65 .

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 2 µl of DNA was added to 100 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Transformation of E. coli XL1 blue with P66

Investigator: Jeff, Rosario, Flo, Johanna, Andi

Aim of the experiment: Transformation of E. coli XL1 blue with P66.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 2 µl of DNA was added to 100 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Transformation of E. coli XL1 blue with P67

Investigator: Jeff, Rosario, Johanna, Andi, Flo

Aim of the experiment: Transformation of E. coli XL1 blue with P67.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 2 µl of DNA was added to 100 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Tuesday, May 14th

Analytical digestion of P15 and P19 to P52

Investigator: Florian, Johanna

Aim of the experiment: Analytical digestion and gelelectrophoresis of P15 and P19 to P52 to check the sequencing results.

Procedure:

  • Batch for analytical digestion of P15 and P19 to P52
volume reagent
5 µl Plasmid DNA
5 µl Mastermix
=10 µl TOTAL

Batch of Mastermix

volume reagent
40 µl NEBuffer 4 (10x)
4 µl BSA (100x)
0.2 µl NotI(10 U/µl)
148 µl ddH2O
=200 µl TOTAL
  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.

Gel 1

Gel 2

  • Discussion:
  • Gel 1, Row 1:
lane part plasmid band 1 band 2 result
2 EreA pDEST14 3,25 kb no cut, no restriction sites in pDEST14
3 mCherry(LMU) pSB1C3 2,0 kb 750 bp probably right
4 GFP(LMU)(845 bp) pSB1C3 2,0 kb 750 bp maybe switched with mKate2(LMU)
5 mKate2(LMU)(702 bp) pSB1C3 2,0 kb 900 bp maybe switched with GFP(LMU)
6 35S + Strong Plant + GFP(1775 bp) pSB1C3 2,0 kb 750 bp ?
7 UVR8(1332 bp) pSB1C3 2,0 kb 1,1 kb ?
8 mStrawberry(708 bp) pSB1C3 2,0 kb 1,4 kb ?
9 DREB1C promoter(463 bp) pSB1C3 2,5 kb probably only one cut -> one NotI site mutated
10 Laccase(1587 bp) pSB1C3 2,0 kb 1,3 kb ?
11 SV40(419 bp) pSB1C3 2,0 kb 1,6 kb this could be the laccase
12 cjBlue(696 bp) pSB1C3 2,0 kb 0,5 kb ?
  • Gel 1, Row 2:
lane part plasmid band 1 band 2 result
2 alcA (843 bp) pSB1C3 2,0 kb 0,7 kb ?
3 eforRed (681 bp) pSB1C3 plasmid plasmid bands (coiled, supercoiled, etc.)
4 amilGFP (699 bp) pSB1C3 2,0 kb 0,7 kb right part
5 mCFP (714 bp) pSB1C3 2,0 kb 0,7 kb right part
6 RD29A promoter (1535 bp) pSB1C3 2,0 kb 1,5 kb right part
7 amilCP (669 bp) pSB1C3 2,0 kb 0,7 kb right part
8 CMV promoter (580 bp) pSB1C3 2,0 kb 0,6 kb right part
9 middle linker (24 bp) pMA-BBFR 2,4 kb short insert went through the gel
10 GSAT (108 bp) pMA-BBFR 2,4 kb 0,1 kb right part
11 GFP(720 bp) pSB1A2 ?
12 long linker (36 bp) pMA-BBFR 2,4 kb short insert went through the gel
  • Gel 2:
lane part plasmid band 1 band 2 result
2 SEG linker (108 bp) pMA-BBFR 2,4 kb short insert went through the gel
3 short linker (12 bp) pMA-BBFR 2,4 kb short insert went through the gel
4 FluA (522 bp) pMA-BBFR 2,4 kb 0,5 kb right part
5 mCherry (769 bp) pSB2K3 4,4 kb 0,75 kb right part
6 Cerulean (778 bp) pSB2K3 ?
7 mRFP1 (706 bp) pSB2K3 4,4 kb 0,7 kb right part
8 mOrange (769 bp) pSB2K3 4,4 kb 0,75 kb right part
9 PIF3-20aa (366 bp) pSB1C3 2,0 kb 0,35 kb right part
10 20aa-PIF3 (366 bp) pSB1C3 2,0 kb 0,35 kb two plasmids and two insert can be seen
11 LexA (ca. 6 kb) pSB1C3 > 8 kb only one cut, huge plasmid

Picking of Plasmid P23 (CaMV35S) and Ligation Product F8 + F15 (TEV)

Investigator: Jeff, Florian

Aim of the study: Picking of Plasmid P23 (CaMV35S) and Ligation Product F8 + F15 (TEV) for Miniprep.

Operational sequence:

  • Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)
  • 1 colony of P23 were picked.
  • 7 colonies of F8 + F15 were picked.

Sequencing of P63 & P64

Investigators: Louise

Aim of the experiment: Sequencing of genes for which no prior sequencing results were available.

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:


Label Name Barcode1 Barcode2
p63 EreA FR01002349 FR01002350
p64 EreA FR01002351 FR01002352

Wednesday, May 15th

Preparative digestion of psB1C3-RFP-generator (P60) and another psB1C3 (P8) as well as P64 (EreA in defect psB1C3 (F8)) with XbaI & AgeI

Investigator: Louise,Johanna

Aim of the experiment:Preparative digestion of psB1C3-RFP-generator (P60) and another psB1C3 (P8) as well as P64 (EreA in defect psB1C3 (F8)) with XbaI & AgeI to recover EreA and prepare two new vectors for cloning.

Procedure:

  • Batch for preparative digestion of PCR product and EreB (F2) with XbaI & AgeI.
volume reagent
20 µl Plasmid (P8,P60,P64)
4 µl NEBuffer 4
0.4 µl BSA (100x)
1 µl AgeI-HF
1 µl XbaI
13.6 µl ddH2O
=40 µl TOTAL
  • Incubation for 2.5 h at 37 °C
  • The digested fragments were labelled F17(P8), F18(P60), F19(P64)

PCR, purification and analytical geleletrophoresis of and EreB (P16)

Investigator: Jeff, Louise

Aim of the experiment: PCR of EreB (P16).

Procedure:

Operational sequence:

  • PCR reaction mixture
volume reagent
10 µl 5x OneTaq Standard Reaction Buffer
1 µl 10 mM dNTPs
1 µl 10 µM Forward Primer (O31 (EreB_for))
1 µl 10 µM Reverse Primer (O32 (EreB_rev))
0.25 µL OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl Plasmid DNA (P15; P16)
35.75 µL ddH2O Water
=50 µL TOTAL
  • Mix with pipette
  • The PCR program TMKS was performed after following scheme:
Initial denaturation 94 °C 30 s
30 cycles 94 °C 30 s
52 °C 60 s
68 °C 80 s
Final extension 68 °C 5 min
Hold 4 °C infinite
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
  • 4 µl of the purified PCR products were mixed with 0.4 µl DNA loading buffer (10x) for analytical gelelectrophoresis
  • Analytical gelelectrophoresis was performed at 90 V for 30 min in 1% agarose gel.
1 kbp ladder F16
Fragment-size like expected

TUM13 20130515 PCR P16.png

Purification and ligation of digested products F17, F18, F19

Investigator: Jeff, Louise

Aim of the experiment: Purification and ligation of digested products F17, F18, F19.

Procedure:

  • To purify the digested Fragments the QIAquick PCR Purification Kit was used. The concentration of the purified product was found to be XX ng/µl via Nano drop.

Ligation of F19 + FXX(pSB1C3)

  • Ligation batch for F19+FXX (positive control)
volume reagent
1.35 µl F8 (74.3 ng/µl, 2086 bp)
X µl F14 (5 ng/µl, 1257 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
  • Cycled ligation has been performed after following protocol in a thermocycler:
99 cycles 12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
Hold 16 °C infinite

Analytical digestion and analytical gelelectrophoresis of P90-P97 (TEV protease, XbaI+AgeI) and P97 (CaMV p35S, XbaI+PstI)

Investigator: Jeff

Aim of the experiment: Analytical digestion and analytical gelelectrophoresis of P90-P97 (TEV protease, XbaI+AgeI) and P97 (CaMV p35S, XbaI+PstI).

Procedure:

  • Mastermix for analytical digestion of P90-P96 with XbaI+AgeI-HF:
volume reagent
16 µl NEBuffer 4 (10x)
1.6 µl BSA (100x)
2 µl XbaI (20 U/µl)
2 µl AgeI-HF (20 U/µl)
118.4 µl ddH2O
=140 µl TOTAL
  • Batch for digestion of P90-P96 with XbaI+AgeI-HF:
volume reagent
2.5 µl Plasmid DNA
17.5 µl Mastermix
=20 µl TOTAL
  • Batch for digestion of P90-P96 with XbaI+AgeI-HF:
volume reagent
2.5 µl Plasmid DNA
2 µl NEBuffer 4 (10x)
0.2 µl BSA (100x)
0.25 µl XbaI (20 U/µl)
0.25 µl AgeI-HF (20 U/µl)
14.8 µl ddH2O
=20 µl TOTAL
  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.

Results:

1 kbp ladder DNA ladder Digestion of P90 Digestion of P91 Digestion of P92 Digestion of P93 Digestion of P94 Digestion of P95 Digestion of P96 Digestion of P97
Insert has expected length Insert has expected length Insert has expected length Insert has expected length Corrupt Insert has expected length Insert has expected length Corrupt
  • The inserts for TEV semm to be right in the most cases, but the backbone has mutated and it should be cloned into a new pSB1C3 with new same restriction enzymes (XbaI+AgeI)!
  • The forward sequencing of CaMV p35S is right but the analytical digestion is wrong. Maybe further parts are downstream of the promoter?! Reverse sequencing should be performed for further analysis.

TUM13 20130515 P90-P96 XbaI.png

Thursday, May 16th

Preparative digestion of pSH21 (P61), psB1A2(P41) and P96 (TEV S19V)

Investigator: Jeff, Katrin

Aim of the experiment: Preparative digestion of pSH21 (P61)and psB1A2(P41) with EcoRI and P96 (TEV S19V) with XbaI & AgeI for subsequent ligation.

Procedure:

  • Batch for preparative digestion of pSH21 with EcoRI
volume reagent
20 µl Plasmid DNA P61
4 µl Buffer 4(10x)
1 µl EcoRI (20 U/µl)
15 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of psB1A2 with EcoRI
volume reagent
20 µl Plasmid DNA P41
4 µl Buffer 4(10x)
1 µl EcoRI (20 U/µl)
15 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P96 (TEV S219V) with XbaI & AgeI.
volume reagent
20 µl Plasmid DNA P96
4 µl Buffer 4(10x)
1 µl XbaI (20 U/µl)
1 µl AgeI-HF (20 U/µl)
13.6 µl ddH2O
=40 µl TOTAL
  • Incubation for 3 h at 37 °C.
  • The resulting fragments were named: F23 (P41) and F22 (P61)
  • 4 µl of DNA loading buffer (10x) were added to the reaction batch containing pSH21 (P61) after digestion and were loaded on an 1% agarose gel for preparative gelelectrophoresis.
  • Preparative gelelectrophoresis was performed at 70 V for 90 min.
1 kbp ladder P61 digestion = F23
Fragment-size like expected; band was cut out

TUM13 20130516 P96 XbaI.AgeI P61 EcoRI.png

  • The band had the expected length. They were cut out and purified using the QIAquick Gel Extraction Kit, QIAGEN

Preparative digestion of ereB (P64) with XbaI & AgeI.

Investigator: Jeff, Louise, Katrin

Aim of the experiment:Preparative digestion of EreB (P64) with XbaI & AgeI.

Procedure:

  • Batch for preparative digestion of PCR product and EreB (F2) with XbaI & AgeI.
volume reagent
20 µl Plasmid
4 µl NEBuffer 4
0.4 µl BSA (100x)
1 µl AgeI-HF
1 µl XbaI
13.6 µl ddH2O
=40 µl TOTAL
  • Incubation for 2.5 h at 37 °C
  • The digested fragments were labelled

Dephosphorylation of F23

Investigator: Jeff, Katrin

Aim of the experiment: Dephosphorylation of cut vector psB1A2 to prevent re-ligation

Procedure:

  • the digestion product was purified with QIAquick PCR Purification Kit, Qiagen in order to change the buffer
  • Batch for the dephosphorylation of F23
volume reagent
30 µl Plasmid DNA F23 (2 µg)
4 µl 10X Fast AP buffer
2 µl FastAP Thermosensitive Alkaline

Phosphatase

4 µl ddH2O
=40 µl TOTAL
  • the reaction batch was spun briefly and incubated at 37 °C for 10 minutes
  • the reaction was stopped by heating to 75 °C for 5 minutes

Ligation of pSH21 (F22) with psB1A2(F23), TEV (P96), EreA (P64), pSB1A2(P41)

Investigator: Jeff, Katrin

Aim of the experiment: ligation of pSH21 (F22) with pSB1A2(F23), TEV (F20) with pSB1C3 (F17), EreA (F19)with pSB1C3 (F17) and EreB (F21) with pSB1C3 (F17)

Procedure:

  • Ligation batch for EreB (F21) with pSB1C3 (F17)
volume reagent
1.6 µl F17 (61,4 ng/µl)
15,4  µl F21 (8,3 ng/µl)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL


  • Ligation batch for TEV (F20) with pSB1C3 (F17)
volume reagent
1.6 µl F17 (61,4 ng/µl)
4,1  µl F20 (24,7  ng/µl)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
11,3 µl ddH20
=20 µl TOTAL
  • Ligation batch for pSH21 (F22) with pSB1A2(F23)
volume reagent
0,85  µl F23 (118,1 ng/µl)
2,3  µl F22 (119,0  ng/µl)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
13,85 µl ddH20
=20 µl TOTAL
  • Ligation batch for EreA (F19) with pSB1C3 (F17)
volume reagent
1.6 µl F17 (61,4 ng/µl)
6,4  µl F19 (8,3 ng/µl)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
9  µl ddH20
=20 µl TOTAL


  • Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
  • the ligation was performed at 16 °C overnight

Friday, May 17th

Transformation of E. coli XL1 blue with F17+F19, F17+F20, F17+F21, F22+F23, F17 (negative control), F23 (negative control)

Investigator: Jeff, Katrin

Aim of the experiment: Transformation of ligation products F17+F21 (pSB1C3, EreB), F17+F20 (pSB1C3, TEV), F17+F19 (pSB1C3, EreA), F23+F22 (pSB1A2, pSH21) and negative controls

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 5 µl of DNA was added to 100 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on chloramphenicol plates (F17+F19, F17+F20, F17+F21, F17 negative) or ampicillin plates (F23+F22, F23 negative control)
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was discarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on chloramphenicol plates (F17+F19, F17+F20, F17+F21) or ampicillin plates (F23+F22)

Quick Change mutagenesis to remove mutations found by sequencing in our gene constructs

Investigator: Jeff

Aim of the experiment: removal of forbidden restiction sites from laccase

Operational sequence

PCR general setup


volume reagent
5 µl 10x Pfu Ultra II buffer
1 µl DNA template (p28)
0,5 µl each 1:10 dilution of appropriate Oligos (1: O26 + O27, 2: O28 + O29, 3: O30 + O31, 4: O32 + O33 )
42 µl ddH2O
1 µl dNTP mix
1 µl Pfu Ultra II DNA polymerase (2.5 U / µl)


PCR cycling parameters

Segment Cycles Temperature Time
1 1 95 °C 30 sec
2 12 95 °C 30 s
55 °C 1 min
68 °C 4 min
3 1 4 °C hold

Transformation of E. coli XL1 blue with the 4 Quickchangeproducts using 1: O26 + O27, 2: O28 + O29, 3: O30 + O31, 4: O32 + O33

Investigator: Jeff, Andi, Johanna

Aim of the experiment: Transformation of E. coli XL1 blue with the 4 Quickchangeproducts using 1: O26 + O27, 2: O28 + O29, 3: O30 + O31, 4: O32 + O33

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 5 µl of DNA was added to 100 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on chloramphenicol plates 1: O26 + O27, 2: O28 + O29, 3: O30 + O31, 4: O32 + O33
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was discarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on chloramphenicol plates (1: O26 + O27, 2: O28 + O29, 3: O30 + O31, 4: O32 + O33)

Saturday, May 18th

Picking of transformed Plasmid E. coli XL1 blue with F17+F19, F17+F20, F17+F21, F22+F23

Investigator: Andi, Jeff

Aim of the study: Picking of transformed Plasmid F17+F19, F17+F20, F17+F21, F22+F23 .

Operational sequence:

  • Picking and overnight culture after standard laboratory's protocol. (CamR/AmpR LB-medium)
  • 2 colonies were picked for every Transformation product.

Sunday, May 19th

Miniprep of overnight culture of transformated E. coli XL1 blue with F17+F19, F17+F20, F17+F21, F22+F23

Investigator: Jeff, Andi

Aim of the experiment: Miniprep of overnight culture of transformated E. coli XL1 blue with F17+F19, F17+F20, F17+F21, F22+F23 of 2 clones picked the day before

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analytical digestion and analytical gelelectrophoresis of F17+F19, F17+F20, F17+F21 and F22+F23

Investigator: Jeff, Andi

Aim of the experiment: Analytical digestion and analytical gelelectrophoresis of ligation products F17+F19, F17+F20, F17+F21, F22+F23.

Procedure:

  • Mastermix for analytical digestion of F17+F19, F17+F20, F17+F21 with XbaI+PstI-HF:
volume reagent
14 µl NEBuffer 4 (10x)
1.4 µl BSA (100x)
1.75 µl XbaI (20 U/µl)
1.75 µl AgeI-HF (20 U/µl)
103.6 µl ddH2O
=122.5 µl TOTAL
  • Batch for digestion of F22+F23 with EcoRI-HF:
volume reagent
2.5 µl Plasmid DNA
2 µl NEBuffer 4 (10x)
15.25 µl ddH2O
0.25 µl EcoRI-HF (20 U/µl)
=20 µl TOTAL
  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed two times for each ligation product at 90 V for 60 min.

Results:

1 kbp ladder DNA ladder Digestion of F17+F19 Digestion of F17+F19 Digestion of F17+F20 Digestion of F17+F20 Digestion of F17+F21 Digestion of F17+F21 Digestion of F22+F23 Digestion of F22+F23
Insert has expected length Insert has expected length, the first line represents the rest of the uncut Plasmid Insert has expected length Insert has expected length, the first line represents the rest of the uncut Plasmid Insert has expected length Insert has expected length Insert has expected length, insert and cut Plasmid overlay each other, because of the same length Corrupt

TUM13 20130519 P98-P103 XbaI.PstI P104-P105 EcoRI(1).png

Quick Change mutagenesis to remove forbidden restriction sites of P28 (Laccase), P100 (TEV S219V), P102 (EreB)

Investigator: Jeff, Andi

Aim of the experiment: Removal of forbidden restiction sites from P28 (Laccase), P100 (TEV S219V), P102 (EreB).

Operational sequence

PCR general setup

Procedure:

PCR

Reaction batch 1

volume reagent
2.5 µl 10x Pfu Ultra II buffer
4 µl Plasmid template (P28/P100/P102)
1 µl 1:10 dilution of O26 (for P28, 10 µM)/O16 (for P102, 10 µM)/O36 (for P100, 10 µM)
16.5 µl ddH2O
0.5 µl dNTP mix
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)

Reaction batch 2

volume reagent
2.5 µl 10x Pfu Ultra II buffer
4 µl Plasmid P38 template
1 µl 1:10 dilution of O27 (for P28, 10 µM)/O17 (for P102, 10 µM)/O37 (for P100, 10 µM)
16.5 µl ddH2O
0.5 µl dNTP mix
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment Cycles Temperature Time
1 1 95 °C 30 s
2 10 95 °C 30 s
55 °C 1 min
68 °C 4 min
  • Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied, but the amount of cycles have been increased to 20.
  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

Analytical gelelectrophoresis of QuickChange products of P100, P102 and P28 to check whether the QuickChange PCR and DpnI digestion were successful

Investigator:

Aim of the experiment:

Procedure:

TUM13 20130519 P28QC P100QC P102QC.png

Transformation of the Quickchange products with P28, P100 and P102 into E. coli XL1 blue

Investigator: Jeff, Andi

Aim of the experiment: Transformation of E. coli XL1 blue with P28, P102 and P100.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 2 µl of DNA was added to 100 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Week 5

Tuesday, May 21st

Quick Change mutagenesis to remove forbidden restriction sites - SDMI - of P28 (Laccase), P100 (TEV S219V), P102 (EreB)

Investigator: Jeff, Andi, Johanna

Aim of the experiment: Removal of forbidden restiction sites from P28 (Laccase), P100 (TEV S219V), P102 (EreB).

Operational sequence

PCR general setup

Procedure:

PCR

Reaction batch 1

volume reagent
2.5 µl 10x Pfu Ultra II buffer
4 µl Plasmid template (P28/P100/P102)
1 µl 1:10 dilution of O26 (for P28, 10 µM)/O16 (for P102, 10 µM)/O36 (for P100, 10 µM)
16.5 µl ddH2O
0.5 µl dNTP mix
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)

Reaction batch 2

volume reagent
2.5 µl 10x Pfu Ultra II buffer
4 µl Plasmid P38 template
1 µl 1:10 dilution of O27 (for P28, 10 µM)/O17 (for P102, 10 µM)/O37 (for P100, 10 µM)
16.5 µl ddH2O
0.5 µl dNTP mix
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment Cycles Temperature Time
1 1 95 °C 30 s
2 10 95 °C 30 s
55 °C 1 min
68 °C 4 min
  • Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied, but the amount of cycles have been increased to 20.
  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

Analytical gelelectrophoresis of QuickChange products of P100, P102 and P28 to check whether the QuickChange PCR and DpnI digestion were successful

Investigator:

Aim of the experiment:

Procedure:

TUM13 20130521 P28QC P100QC P102QC.png

Transformation of the Quickchange products with P28, P100 and P102 into E. coli XL1 blue

Investigator: Jeff, Andi, Johanna

Aim of the experiment: Transformation of E. coli XL1 blue with P28, P102 and P100.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 2 µl of DNA was added to 100 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Sequencing of P105, P28, P100, P102 & P98

Investigators: Jeff, Andi,Johanna

Aim of the experiment: Sequencing of genes for which no prior sequencing results were available.

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label Name Barcode1 Barcode2
p105 IRES FR01002288 FR01002269
p102 EreB FR01002270 FR01002275
p100 TEV S219V FR01002278 FR01002279
p98 EreA FR01002276 FR01002277
p28 Laccase FR01002280

Preparation of Knop-Medium for Moss

Investigator: Jeff, Andi, Johanna, Rosario

Aim of the experiment: Preparation of Knop-Medium for Moss

Procedure:

  • All four prepared stocks have been: 25g/L KH2PO4; 25g/L KCl; 25g/L MgSO4 x 7 H2O; 100g/L Ca(NO3)2
  • Volume of prepared Medium: 1L
  • 10 ml of each stock were converted into a 1,8L Erlenmeyer flask and filled up to 1L with destilled water (ELGA); 12,5mg FeSO4 x 7 H2O were added; the pH was set to 5.8 with KOH
  • The prepared Volume of 1L was shared equally to 500ml Erlenmeyer flasks, so each Erlenmeyer flask contained 200 ml of the Knop-Medium
  • All flasks have been autoclaved

Picking of transformed Plasmid E. coli XL1 blue with QuickChange products of P100 (TEV, QC with O36/O37) and P102 (EreB, QC with O16/O17)

Investigator: Andi, Jeff, Johanna, Rosario

Aim of the study: Picking of transformed Plasmid E. coli XL1 blue with QuickChange products of P100 (TEV, QC with O36/O37) and P102 (EreB, QC with O16/O17) from Sunday, May 19th.

Operational sequence:

  • Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)
  • 3 colonies for P100QC (TEV, QC with O36/O37) and 5 colonies from P102QC (EreB, QC with O16/O17) were picked from the plates.

Wednesday, May 22nd

Miniprep of overnight culture of transformated E. coli XL1 blue with QuickChange products of P100 (TEV, QC with O36/O37) and P102 (EreB, QC with O16/O17)

Investigator: Jeff

Aim of the experiment: Miniprep of overnight culture of transformated E. coli XL1 blue with QuickChange products of P100 (TEV, QC with O36/O37) and P102 (EreB, QC with O16/O17).

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Picking of transformed Plasmid E. coli XL1 blue with QuickChange products of P28 (Laccase, QC with O26/O27)

Investigator: Jeff

Aim of the study: Picking of transformed Plasmid E. coli XL1 blue with QuickChange products of P28 (Laccase, QC with O26/O27).

Operational sequence:

  • Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)
  • 8 colonies for P28QC (Laccase, QC with O26/O27) were picked from the plates.

Analytical digestion and analytical gelelectrophoresis of P106, P107, P108, P109, P110, P111, P112, P113

Investigator: Jeff, Rosario

Aim of the experiment: Analytical digestion and analytical gelelectrophoresis of Quickchanges to check whether the transformation of E. coli Xl1 Blue with QuickChanges products of P100 (TEV, QC with O36/O37: P111, P112, P113) and P102 (EreB, QC with O16/O17: P106, P107, P108, P109, P110) were successful

Procedure:


Mastermix for analytical digestion of P106, P107, P108, P109, P110, P111, P112, P113 with AgeI-HF and XbaI:

volume reagent
20 µl NEBuffer 4 (10x)
2 µl BSA (100x)
2.5 µl AgeI-HF (20 U/µl)
2.5 µl XbaI
148 µl ddH2O
= 175 µl TOTAL
  • 17,5 µl Mastermix with 2,5 µl Plasmid


Mastermix for analytical digestion of P111, P112, P113 with XbaI and SpeI and P106, P107, P108, P109, P110 with XbaI and PstI-HF:

volume reagent
20 µl NEBuffer 4 (10x)
2 µl BSA (100x)
2.5 µl XbaI
148 µl ddH2O
= 172,5 µl TOTAL
  • 17,25 µl Mastermix with 2,5 µl Plasmid and 0,25 µl SpeI or PstI respectively


Mastermix for analytical digestion of with P106, P107, P108, P109, P110 EcoRI and SpeI:

volume reagent
12 µl NEBuffer 4 (10x)
1,2 µl BSA (100x)
1,5 µl EcoRI-HF
1,5 µl SpeI-HF
88.8 µl ddH2O
= 105 µl TOTAL
  • 17,5 µl Mastermix with 2,5 µl Plasmid


Mastermix for analytical digestion of with P106, P107, P108, P109, P110 XbaI and SpeI:

volume reagent
12 µl NEBuffer 4 (10x)
1,2 µl BSA (100x)
1,5 µl XbaI
1,5 µl SpeI-HF
88.8 µl ddH2O
= 105 µl TOTAL
  • 17,5 µl Mastermix with 2,5 µl Plasmid


  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.


Results:


TUM13 20130522 P111-P113 XbaI.AgeI P111-P113 XbaI.SpeI.png

1 kbp ladder DNA ladder P111 XbaI + AgeI P112 XbaI + AgeI P113 XbaI + AgeI P111 XbaI + SpeI P112 XbaI + SpeI P113 XbaI + SpeI
Ctrl Ctrl Ctrl QC succesful QC failed QC succesful


TUM13 20130522 P106-P110 XbaI.AgeI P106-P110 XbaI.PstI.png

1 kbp ladder DNA ladder P106 XbaI + AgeI P107 XbaI + AgeI P108 XbaI + AgeI P109 XbaI + AgeI P110 XbaI + AgeI P106 XbaI + PstI P107 XbaI + PstI P108 XbaI + PstI P109 XbaI + PstI P110 XbaI + PstI
Ctrl Ctrl Ctrl Ctrl Ctrl PstI inside as should be; useless experiment PstI inside as should be; useless experiment PstI inside as should be; useless experiment PstI inside as should be; useless experiment PstI inside as should be; useless experiment


TUM13 20130522 P106-P110 XbaI.SpeI P106-P110 EcoRI.SpeI.png

1 kbp ladder DNA ladder P106 XbaI + SpeI P107 XbaI + SpeI P108 XbaI + SpeI P109 XbaI + SpeI P110 XbaI + SpeI P106 EcoRI + SpeI P107 EcoRI + SpeI P108 EcoRI + SpeI P109 EcoRI + SpeI P110 EcoRI + SpeI
Ctrl Ctrl Ctrl Ctrl Ctrl QC succesful QC succesful QC succesful QC succesful QC failed

Thursday, May 23rd

Sequencing of P111, P113 and P109 (?)

Investigator: Jeff

Aim of the experiment: Sequencing of P111, P113 and P109 to check whether the QuickChange brought further unwanted mutations.

Procedure:

PCR of P111 (TEV S219V, SpeI-less) to generate Split-TEV

Investigator: Jeff

Aim of the experiment: PCR of P111 (TEV S219V, SpeI-less) to generate Split-TEV. To generate N-TEV, the primer O38 and O39 were used. To generate C-TEV, the primer O40 and O41 were used.

Procedure:

Operational sequence:

  • PCR reaction mixture
volume reagent
10 µl 5x OneTaq Standard Reaction Buffer
1 µl 10 mM dNTPs
1 µl 10 µM Forward Primer (O38 (N_Split_TEV_fw) for generating N-TEV; O40 (C_Split_TEV_fw) for generating C-TEV)
1 µl 10 µM Reverse Primer (O39 (N_Split_TEV_rv) for generating N-TEV; O41 (C_Split_TEV_rv) for generating C-TEV)
0.25 µL OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl Plasmid DNA (P111)
35.75 µL ddH2O Water
=50 µL TOTAL
  • Mix with pipette
  • The PCR program was performed after following scheme with following conditions:
Initial denaturation 94 °C 30 s
30 cycles 94 °C 30 s
51 °C 60 s
68 °C 30 s
Final extension 68 °C 5 min
Hold 4 °C infinite
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
  • Resulting product was labeled as F24 (N-TEV) and F25 (C-TEV).

Analytical gelelectrophoresis of F24 and F25 (N-TEV and C-TEV)

Investigator: Jeff, Rosario

Aim of the experiment: Analytical gelelectrophoresis of F24 and F25 (N-TEV and C-TEV) to check whether PCR has worked.

Procedure:

  • 4 µl of F24 and F25 were mixed with seperately with 0.44 µl of DNA loading buffer (10x) and analytical gelelectrophoresis was performed at 1% agarose gel for 60 min at 90 V.

TUM13 20130523 F24 F25.png

  • PCR products has expected length, but there are unspecific products. So we have to be cautious and have to check if we ligate the right products.

Preparative digestion of PCR products (N-TEV and C-TEV) of P111 (TEV S219V, SpeI-less) with XbaI & AgeI

Investigator: Jeff, Rosario

Aim of the experiment: Preparative digestion of PCR products (N-TEV and C-TEV) of P111 (TEV S219V, SpeI-less) with XbaI & AgeI to clone it into pSB1C3.

Procedure:

  • Batch for preparative digestion of PCR products (N-TEV and C-TEV) of P111 (TEV S219V, SpeI-less) with XbaI & AgeI.
volume reagent
25 µl PCR product F24/F25
5 µl NEBuffer 4
0.5 µl BSA (100x)
1 µl AgeI-HF
1 µl XbaI
17.5 µl ddH2O
=50 µl TOTAL
  • Incubation for 150 min at 37 °C.
  • Digested products were purified with QIAquick PCR Purification Kit, QIAGEN.
  • The digested fragments were labelled F26 (digestion of F24 with XbaI&AgeI) and F27 (digestion of F25 with XbaI&AgeI)

Ligation of F17+F26 (N-TEV in pSB1C3) and F17+F27 (C-TEV in pSB1C3)

Investigator: Jeff, Rosario

Aim of the experiment: Ligation of F17+F26 (N-TEV in pSB1C3) and F17+F27 (C-TEV in pSB1C3).

Procedure:

  • Ligation batch for F17+F26:
volume reagent
1.63 µl F17 (61.4 ng/µl, 2086 bp)
3.20 µl F26 (15.9 ng/µl, 354 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
12.17 µl ddH2O
=20 µl TOTAL
  • Ligation batch for F17+F27 (positive control)
volume reagent
1.63 µl F17 (61.4 ng/µl, 2086 bp)
2.41 µl F27 (21.1 ng/µl, 354 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
12.96 µl ddH2O
=20 µl TOTAL
  • Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
  • Cycled ligation has been performed after following protocol in a thermocycler:
99 cycles 12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
Hold 16 °C infinite
  • Lid temperature = 37 °C

Miniprep of overnight culture of transformated E. coli XL1 blue with the first Quickchange (SDMI) Product of P28

Investigator: Jeff, Andi, Rosario, Johanna

Aim of the experiment: Miniprep of overnight culture of transformated E. coli XL1 blue with the first Quickchange Product of P28. 8 clones were picked the day before.

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analytical digestion and analytical gelelectrophoresis of P28, P117, P118, P119, P120, P121, P122, P123

Investigator: Rosario, Andi, Johanna, Jeff

Aim of the experiment: Analytical digestion and analytical gelelectrophoresis of P28 (promoter SV40) and quickchange products P117, P118, P119, P120, P121, P122, P123 (all QC - SDMI - of Bpul Laccase to remove forbidden AgeI).

Procedure:

  • Mastermix for analytical digestion of P28, P117, P118, P119, P120, P121, P122, P123 AgeI-HF:
volume reagent
20 µl NEBuffer 4 (10x)
2 µl BSA (100x)
2.5 µl AgeI-HF (20 U/µl)
150.5 µl ddH2O
=175 µl TOTAL
  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed two times for each ligation product at 90 V for 60 min.

Results:

TUM13 20130523 P28 AgeI P116-P123 AgeI.png

1 kbp ladder DNA ladder Digestion of P28 Digestion of P116 Digestion of P117 Digestion of P118 Digestion of P119 Digestion of P120 Digestion of P121 Digestion of P122 Digestion of P123
as expected QC successful QC successful QC successful QC successful QC successful QC successful QC successful QC successful

Quick Change mutagenesis to remove forbidden restriction sites - SDMII - of P28 (Laccase), P106 (EreB)

Investigator: Jeff, Andi, Johanna, Rosario

Aim of the experiment: Removal of forbidden restiction sites from P28 (Laccase), P106 (EreB).

Procedure: Quickchange - PCR

Reaction batch - P106

volume reagent Additional information
5 µl 10x Pfu Ultra II buffer
2 µl Plasmid template (P106 - 1:10 dilution) need to get to 50 ng/µl
1.25 µl 1:10 dilution of O18 (for P106, 10 µM)
1.25 µl 1:10 dilution of O19 (for P106, 10 µM)
38.5 µl ddH2O
1 µl dNTP mix
1 µl Pfu Ultra II DNA polymerase (2.5 U / µl)


PCR cycling parameters - P106

Segment Cycles Temperature Time
1 1 95 °C 30 s
2 18 95 °C 30 s
52 °C 1 min
68 °C 4 min
  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.


Reaction batch - P116 (Laccase)

volume reagent Additional information
5 µl 10x Pfu Ultra II buffer
2 µl Plasmid template - 1:5 dilution need to get to 50 ng/µl
1.25 µl 1:10 dilution of O28 for P116
1.25 µl 1:10 dilution of O29 for P116
38.5 µl ddH2O
1 µl dNTP mix
1 µl Pfu Ultra II DNA polymerase (2.5 U / µl)


PCR cycling parameters - P116

Segment Cycles Temperature Time
1 1 95 °C 30 s
2 18 95 °C 30 s
52 °C 1 min
68 °C 4 min
  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

Transformation of the Quickchange products with P28, P100 and P102 into E. coli XL1 blue

Investigator: Jeff, Andi, Johanna, Rosario

Aim of the experiment: Transformation of E. coli XL1 blue with Quickchangeproduct SDM II of P106 and P116.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 2 µl of DNA was added to 100 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Friday, May 24th

Transformation of E. coli XL1 blue with ligation products F17+F26 (N-TEV S219V in pSB1C3) and F17+F27 (C-TEV S219V in pSB1C3)

Investigator: Jeff, Florian

Aim of the experiment: Transformation of E. coli XL1 blue with ligation products F17+F26 (N-TEV S219V in pSB1C3) and F17+F27 (C-TEV S219V in pSB1C3).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA was added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions was plated again on a new chlorampenicol plates.

Quick Change mutagenesis to remove forbidden restriction sites - SDMII - of P116 (Laccase), P109 (EreB)

Investigator: Volker, Louise, Katrin, Flo

Aim of the experiment: Removal of forbidden restiction sites from P116 (Laccase), P109 (EreB).

Procedure: Quickchange - PCR

Reaction batch - P109

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid template (P109 - 1:10 dilution) need to get to 50 ng/µl
1 µl 1:10 dilution of O18 (for P109, 10 µM)
1 µl 1:10 dilution of O19 (for P109, 10 µM)
18 µl ddH2O
1 µl dNTP mix 50mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)


PCR cycling parameters - P109

Segment Cycles Temperature Time
1 1 95 °C 30 s
2 21 95 °C 30 s
52 °C 1 min
68 °C 4 min 50 s
  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.


Reaction batch - P116 (Laccase)

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid template - 1:5 dilution need to get to 50 ng/µl
1 µl 1:10 dilution of O28 for P116
1 µl 1:10 dilution of O29 for P116
18.0 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)


PCR cycling parameters - P116

Segment Cycles Temperature Time
1 1 95 °C 30 s
2 21 95 °C 30 s
52 °C 1 min
68 °C 4 min 50 s
  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

Transformation of the Quickchange products into E. coli XL1 blue

Investigator: Volker

Aim of the experiment: Transformation of E. coli XL1 blue with Quickchangeproduct SDM II of P109 and P116.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 2 µl of DNA was added to 100 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Saturday, May 25th

Analytical gelelectrophoresis of QuickChange products of P116 and P109 to check whether the QuickChange PCR and DpnI digestion were successful

Investigator: Volker, Louise, Katrin, Flo

Aim of the experiment: Analytical gelelectrophoresis of QuickChange products of P116 and P109 to check whether the QuickChange PCR and DpnI digestion were successful

Procedure:

  • 5 µl of the PCR Quickchange products were either mixed with with 0.5 µl of DNA loading buffer (10x) and analytical gelelectrophoresis was performed at 1% agarose gel for 60 min at 90 V.


Picking of transformed Plasmid E. coli XL1 blue with QuickChange products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19)

Investigator: Volker

Aim of the study: Picking of transformed Plasmid E. coli XL1 blue with QuickChange products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19).

Procedure:

  • Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)
  • 5 colonies for P116QC (Laccase, QC with O28/O29) and 6 colonies for P109QC (EreB, QC with O18/O19) were picked from the plates.

Picking of E. coli XL1 blue with ligation products F17+F26 (N-TEV S219V in pSB1C3) and F17+F27 (C-TEV S219V in pSB1C3)

Investigator: Volker

Aim of the study: Picking of E. coli XL1 blue with ligation products F17+F26 (N-TEV S219V in pSB1C3) and F17+F27 (C-TEV S219V in pSB1C3).

Procedure:

  • Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)
  • 5 colonies were picked from plates for E. colis XL1 blue transformed with ligation products F17+F26 (N-TEV S219V in pSB1C3) and F17+F27 (C-TEV S219V in pSB1C3).

Sunday, May 26th

Miniprep of overnight cultures of transformated E. coli XL1 blue with QuickChange products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19)

Investigator: Katrin, Louise

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with QuickChange products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19).

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analytical digestion and gelelectrophoresis to check whether the transformation of E. coli Xl1 Blue with QuickChanges products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19) were successful

Investigator: Katrin, Louise

Aim of the experiment:Analytical digestion and gelelectrophoresis to check whether the transformation of E. coli Xl1 Blue with QuickChanges products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19)

Procedure:

  • analytical digestion of Quickchange products of P109 (EreB, QC with O18/O19)
volume reagent
2 µl NEB CutSmart Buffer (10x)
0,25  µl PstI-HF (20 U/µl)
2,5 µl DNA from Miniprep
15,25 µl ddH2O
=20 µl TOTAL
  • in order to check if the QC was successful, P109 (before QC) was also digested


  • analytical digestion of Quickchange products of P116 (Laccase, QC with O28/O29)
volume reagent
2 µl NEB CutSmart Buffer (10x)
0,25 µl EcoRI-HF
0,25  µl NgoMIV
2,5 µl DNA from Miniprep
15  µl ddH2O
=20 µl TOTAL
  • in order to check if the QC was successful, P116 (before QC) was also digested
  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.


TUM13 20130526 anal Verd P116 EcoRI.NgoMIV P128-P131,P124 EcoRI.NgoMIV P126,P137-P139 XbaI.AgeI.png

1 kbp ladder DNA ladder# P116 (Laccase before QCII) digested with EcoRI-HF, NgoMIV Laccase after QCII (P128) digested with EcoRI-HF, NgoMIV Laccase after QCII (P129) digested with EcoRI-HF, NgoMIV Laccase after QCII (P130) digested with EcoRI-HF, NgoMIV Laccase after QCII (P131) digested with EcoRI-HF, NgoMIV Laccase after QCII (P124) digested with EcoRI-HF, NgoMIV belongs to other experiment belongs to other experiment belongs to other experiment belongs to other experiment
as expected as expected as expected as expected as expected as expected

Laccase P124 was chosen for sequencing


TUM13 20130526 anal Verd PP127,P140-P142 XbaI.AgeI P125,P132-P136 PstI P109 PstI.png

1 kbp ladder DNA ladder belongs to other experiment belongs to other experiment belongs to other experiment belongs to other experiment EreB after QCII (P125) digested with PstI EreB after QCII (P132) digested with PstI EreB after QCII (P133) digested with PstI EreB after QCII (P134) digested with PstI EreB after QCII (P135) digested with PstI EreB after QCII (P136) digested with PstI EreB before QCII (P109) digested with PstI
as expected as expected as expected as expected as expected as expected as expected

EreB P125 was chosen for sequencing

Miniprep of ligation products of N-TEV (F17+F26) and C-TEV (F17+F25) with pSB1C3

Investigator: Katrin, Louise

Aim of the experiment: Miniprep of ligation products of N-TEV (F17+F26) and C-TEV (F17+F25) with pSB1C3

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analytical digestion and gelelectrophoresis of ligation products F17 + F26, F17 + F27

Investigator: Katrin, Louise

Aim of the experiment: Analytical digestion and gelelectrophoresis to check whether the ligation of N-TEV (F17+F26) and C-TEV (F17+F25) in pSB1C3 were successful

Procedure:

  • analytical digestion of ligation products of N-TEV (F17+F26) and C-TEV (F17+F25)in pSB1C3

Mastermix:

volume reagent
22 µl NEB Buffer 4 (10x)
2,2  µl BSA (100X)
2,75 µl XbaI
2,75 µl AgeI-HF
162,8 µl ddH2O
=192,5 µl TOTAL


  • 2,5 µl of miniprep products were added to 17,5  µl Mastermix
  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.


TUM13 20130526 anal Verd P116 EcoRI.NgoMIV P128-P131,P124 EcoRI.NgoMIV P126,P137-P139 XbaI.AgeI.png

1 kbp ladder DNA ladder# belongs to other experiment belongs to other experiment belongs to other experiment belongs to other experiment belongs to other experiment belongs to other experiment N-TEV ligated into pSB1C3 (F17 + F26) (P126) digested with XbaI, AgeI-HF N-TEV ligated into pSB1C3 (F17 + F26) (P137) digested with XbaI, AgeI-HF N-TEV ligated into pSB1C3 (F17 + F26) (P138) digested with XbaI, AgeI-HF N-TEV ligated into pSB1C3 (F17 + F26) (P139) digested with XbaI, AgeI-HF
as expected as expected as expected as expected

N-TEV ligated into pSB1C3 (F17 + F26) (P126) was chosen for sequencing


TUM13 20130526 anal Verd PP127,P140-P142 XbaI.AgeI P125,P132-P136 PstI P109 PstI.png

1 kbp ladder DNA ladder C-TEV ligated into pSB1C3 (F17 + F27) (P127) digested with XbaI, AgeI-HF C-TEV ligated into pSB1C3 (F17 + F27) (P140) digested with XbaI, AgeI-HF C-TEV ligated into pSB1C3 (F17 + F27) (P141) digested with XbaI, AgeI-HF C-TEV ligated into pSB1C3 (F17 + F27) (P142) digested with XbaI, AgeI-HF belongs to other experiment belongs to other experiment belongs to other experiment belongs to other experiment belongs to other experiment belongs to other experiment belongs to other experiment
as expected as expected as expected as expected

C-TEV ligated into pSB1C3 (F17 + F27) (P127) was chosen for sequencing

Sequencing of P124-P127

Investigators: Louise, Katrin

Aim of the experiment: Sequencing of QCII products (EreB, Laccase) and ligation product of split-TEV

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label Name Barcode1 Barcode2
p124 Miniprep of transformation of QC product of P116 (Laccase, QC with O28/O29)(QC - SDMII - of Bpul Laccase to remove forbidden NgoMIV) FR01002337 (VR) FR01002338 (VF2)
p125 Miniprep of transformation of QC product of P109 (EreB, QC with O18/O19) (QC - SDMII - of EreB to remove forbidden PstI) FR01002339 (VR) FR01002340 (VF2)
p126 Miniprep of ligation product F17 + F26 (N-TEV) FR01002341 (VR) FR01002342 (VF2)
p127 Miniprep of ligation product F17 + F27 (C-TEV) FR01002343 (VR) FR01002344 (VF2)

Week 6

Monday, May 27th

Oligohybridization of MiniMCS (O56,O57) and Spytag (O54,O55)

Investigator: Johanna

Aim of the experiment: Oligohybridization of MiniMCS (MiniMCSII_fw (O56) and MiniMCSII_rv (O57) and of Spytag (SpyTag_fw (O54) and SpyTag_rv (O55))

Operational sequence

  • 25 µL of 100 pmol/µl of MiniMCS fw and 25 µL of 100 pmol/µl MiniMCS rev in one Eppi. 25 µL of 100 pmol/µl of Spytag fw and 25 µL of 100 pmol/µl Spytag rev in a second Eppi
  • Heating up to 95 °C for 5 min
  • Cooling at RT in a styropor box overnight

PCR of npt-Kasette (P115), PIF3 (P51), pActin (P114) and t35S (P114)

Investigator: Johanna

Aim of the experiment: PCR of npt-Kasette (P115), PIF3 (P51), pActin (P114) and t35S (P114).

Procedure:

Operational sequence:

  • PCR reaction mixture
volume reagent
10 µl 5x OneTaq Reaction GC Buffer
1 µl 10 mM dNTPs
1 µl 10 µM Forward Primer (P114:O62 (pActin_for); P114:O60 (t35S_for); P115:O58 (npt-Kasette_for); P51:O66 (PIF3_for))
1 µl 10 µM Reverse Primer (P114:O63 (pActin_rev); P114:O61 (t35S_rev); P115:O59 (npt-Kasette_rev); P51:O67 (PIF3_rev))
0.25 µL OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl Plasmid DNA (P114; P115; P51)
35.75 µL ddH2O Water
=50 µL TOTAL
  • Mix with pipette
  • The PCR program was performed after following scheme:
Initial denaturation 94 °C 30 s
30 cycles 94 °C 30 s
49 °C 60 s
68 °C 100 s
Final extension 68 °C 5 min
Hold 4 °C infinite
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

Results:

  • PCR did not work properly because primer concentration was wrong.

Preparative digestion and gelelectrophoresis of P45 (FluA-Psb1C3) with XbaI & AgeI, P39 with AgeI & PstI, P20 with NgoMIV & PstI

Investigator: Andi

Aim of the experiment:Preparative digestion of P45 (FluA-Psb1C3) with XbaI & AgeI, P39 with AgeI & PstI, P20 with NgoMIV & PstI to clone fragment of P45 into F17 (pSB1C3) and to fusion Middle linker of P39 with GFP of P20.

Procedure:

Batch for preparative digestion of P45 (FluA-Psb1C3) with XbaI & AgeIwith XbaI & AgeI.

volume reagent
20 µl P45
4 µl NEBuffer 4
0.4 µl BSA (100x)
2 µl AgeI-HF
2 µl XbaI
11.6 µl ddH2O
=40 µl TOTAL

Batch for preparative digestion of P39 (Middle-Linker) with PstI and AgeI.

volume reagent
20 µl P39
4 µl NEBuffer 4
0.4 µl BSA (100x)
2 µl AgeI-HF
2 µl PstI
11.6 µl ddH2O
=40 µl TOTAL

Batch for preparative digestion of P20 (GFP-Psb1C3) with NgoMIV and PstI.

volume reagent
20 µl P45
4 µl NEBuffer 4
0.4 µl BSA (100x)
2 µl NgoMIV
2 µl PstI
11.6 µl ddH2O
=40 µl TOTAL
  • Incubation for 120 min at 37 °C.
  • Preparative gelelectrophoresis was performed at 90 V for 1.5 h.

TUM13 20130527 P45 XbaI.AgeI P39 AgeI.PstI P20 NgoMIV.PstI.png

1 kbp ladder P20 digested with NgoMIV&PstI (=F30) P20 digested with NgoMIV&PstI (=F30) P39 digested with AgeI&PstI (=F29) P39 digested with AgeI&PstI (=F29) P45 digested with XbaI&AgeI (=F28) P45 digested with XbaI&AgeI (=F28)
lower band was cut out lower band was cut out band was cut out band was cut out lower band was cut out lower band was cut out
  • Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.

Quick Change mutagenesis to remove forbidden restriction sites - SDMIII - of P124(Laccase), P125 (EreB)

Investigator: Andi

Aim of the experiment: Removal of forbidden restiction sites from P124 (Laccase), P125 (EreB).

Procedure: Quickchange - PCR

Reaction batch - P124

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid template (P124 - 1:6 dilution) need to get to 50 ng/µl
1 µl 1:10 dilution of O30 (for P125, 10 µM)
1 µl 1:10 dilution of O31 (for P125, 10 µM)
18 µl ddH2O
1 µl dNTP mix 50mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)

Reaction batch - P125

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid template (P125 - 1:6 dilution) need to get to 50 ng/µl
1 µl 1:10 dilution of O20 (for P125, 10 µM)
1 µl 1:10 dilution of O21 (for P125, 10 µM)
18 µl ddH2O
1 µl dNTP mix 50mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters for both batches

Segment Cycles Temperature Time
1 1 95 °C 30 s
2 21 95 °C 30 s
52 °C 1 min
68 °C 4 min 50 s
  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

Preparation of ordered primers

Investigators: Andi

Aim of the experiment: Dissolving of primer pallets to provide a concentration of 100 pmol/µl

Procedure: We added an amount of water which gave us a final primer concentration of 100 pmol/µl.

Primers were labeled as follows:

Label Oligonr.
O54 Spytag_fw
O55 Spytag_rev
O56 MiniMCSII_fw
O57 MiniMCSII_rev
O58 Npt_for
O59 Npt_rev
O60 t35S_for
O61 t35S_rev
O62 pACT_for
O63 pACT_rev
O64 NucA_for
O65 NucA_rev
O66 Pif3_for
O67 Pif3_rev

Transformation of P45, P51, P100 and P102 into E. coli XL1 blue

Investigator: Andi, Johanna

Aim of the experiment: Transformation of E. coli XL1 blue with P45, P51, P102 and P100.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 2 µl of DNA was added to 100 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Picking of transformed Plasmid E. coli XL1 blue with P114, P115

Investigator: Andi

Aim of the study: Picking of transformed Plasmid E. coli XL1 blue with P114, P115.

Procedure:

  • Picking and overnight culture after standard laboratory's protocol. (AmpR LB-medium)
  • 5 colonies of P114 and P115 were picked from the plates.

Ligation of F29+F30 (GFP in pSB1C3 after middle linker, ( Gly-Gly-Ser-Gly)x2) and F17+F27 (FluA in pSB1C3)

Investigator: Jeff

Aim of the experiment: Ligation of F29+F30 (GFP in pSB1C3 after middle linker, ( Gly-Gly-Ser-Gly)x2) and F17+F27 (FluA in pSB1C3).

Procedure:

  • Ligation batch for F29+F30:
volume reagent
4.22 µl F29 (23.7 ng/µl, 2110 bp)
10.48 µl F30 (9.5 ng/µl, ~700 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
2.3 µl ddH2O
=20 µl TOTAL
  • Ligation batch for F17+F28:
volume reagent
1.63 µl F17 (61.4 ng/µl, 2086 bp)
7.8 µl F28 (9.6 ng/µl, 522 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
7.57 µl ddH2O
=20 µl TOTAL
  • Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
  • Cycled ligation has been performed after following protocol in a thermocycler:
99 cycles 12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
Hold 16 °C infinite
  • Lid temperature = 37 °C

Tuesday, May 28th

Analytical gelelectrophoresis of PCR products of P114 (O62&O63), P114 (O60&O61), P115 (O58&O59), P51 (O66&O67)

Investigator: Jeff, Rosario

Aim of the experiment: Analytical gelelectrophoresis of PCR products of P114 (O62&O63), P114 (O60&O61), P115 (O58&O59), P51 (O66&O67).

Procedure:

  • 4 µl of PCR products of P114 (O62&O63), P114 (O60&O61), P115 (O58&O59), P51 (O66&O67) were mixed with seperately with 0.44 µl of DNA loading buffer (10x) and analytical gelelectrophoresis was performed at 1% agarose gel for 40 min at 90 V.

TUM13 20130528 PCR F33 F34 F35 F36.png

1 kbp ladder PCR products of P114 (O62&O63), P114 (O60&O61), P115 (O58&O59), P51 (O66&O67) PCR products of P114 (O60&O61) PCR products of P115 (O58&O59) PCR products of P51 (O66&O67)
PCR did work, but strong primer dimer signal PCR did work, but strong primer dimer signal PCR did not work PCR did work, but strong primer dimer signal
  • Wrong buffer (GC buffer, instead of standard reaction buffer, was used and 10x too much primers were also used)

Transformation of Quickchangeproduct SDMIII of P124 (Laccase) and P125 (EreB) + ligation product of F29+F30 + ligation product of F17+F19 into E. coli XL1 blue

Investigator: Andi, Johanna

Aim of the experiment: Transformation of Quickchangeproduct SDMIII of P124 (Laccase) and P125 (EreB) + ligation product of F29+F30 + ligation product of F17+F19 + ligation product of F17+NK + ligation product of F29+NK into E. coli XL1 blue.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 2 µl of DNA was added to 100 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Sequencing of P133 and P134

Investigators: Louise, Johanna

Aim of the experiment: Sequencing of two more QCII products (EreB) Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label Name Barcode1 Barcode2
p133 Miniprep of transformation of QC product of P109 (EreB, QC with O18/O19) (QC - SDMII - of EreB to remove forbidden PstI) FR01002287 (fw) FR01002296 (rev)
p134 Miniprep of transformation of QC product of P109 (EreB, QC with O18/O19) (QC - SDMII - of EreB to remove forbidden PstI) FR01002295 (fw) FR01002294 (rev)

Miniprep of overnight cultures of transformated E. coli XL1 blue of P114 and P115

Investigator: Andi

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue of P114 and P115.

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Picking of of E. coli XL1 blue transformed with P45, P51, P100, P102

Investigator: Jeff, Rosario

Aim of the experiment: Picking of of E. coli XL1 blue transformed with P45, P51, P100, P102.

Procedure:

  • Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x) (P51, P100, P102)/4 µL Ampicillin (1000x) (P45).
  • 1 colony for each plasmid were picked.
  • These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight.

PCR of npt-Kasette (P144), PIF3 (P51), pActin (P143) and t35S (P143)

Investigator: Rosario, Jeff

Aim of the experiment: PCR of npt-Kasette (P144), PIF3 (P51), pActin (P143) and t35S (P143).

Procedure:

Operational sequence:

  • PCR reaction mixture
volume reagent
10 µl 5x OneTaq Reaction Standard Reaction Buffer
1 µl 10 mM dNTPs
1 µl 10 µM Forward Primer (P143:O62 (pActin_for); P143:O60 (t35S_for); P144:O58 (npt-Kasette_for); P51:O66 (PIF3_for))
1 µl 10 µM Reverse Primer (P143:O63 (pActin_rev); P143:O61 (t35S_rev); P144:O59 (npt-Kasette_rev); P51:O67 (PIF3_rev))
0.25 µL OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl Plasmid DNA (P114; P115; P51)
35.75 µL ddH2O Water
=50 µL TOTAL
  • Mix with pipette
  • The PCR program was performed after following scheme:
Initial denaturation 94 °C 30 s
30 cycles 94 °C 30 s
47 °C 60 s
68 °C 120 s
Final extension 68 °C 5 min
Hold 4 °C infinite
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

Analytical gelelectrophoresis of F33, F34, F35, F36

Investigator: Jeff, Rosario

Aim of the experiment: Analytical gelelectrophoresis of F33, F34, F35, F36.

Procedure:

  • 4 µl of F33, F34, F35 and F36 were mixed with seperately with 0.44 µl of DNA loading buffer (10x) and analytical gelelectrophoresis was performed at 1% agarose gel for 40 min at 90 V.

TUM13 20130528 PCR F33 F34 F35 F36.png

1 kbp ladder F33 F33 F33 F33
PCR did not work, maybe high GC content? PCR products has expected length, some byproducts PCR products has expected length, some byproducts PCR products has expected length

Wednesday, May 29th

Transformation of E. coli XL1 blue with SDMIII product of P124, ligation product of F29+F30 and ligation product of F17+F18

Investigator: Andi

Aim of the experiment: Transformation of E. coli XL1 blue with SDMIII product of P124, ligation product of F29+F30 and ligation product of F17+F18.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 5 µl of DNA was added to 100 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Miniprep of overnight cultures of transformated E. coli XL1 blue of P100, P102, P51, P45

Investigator: Rosario

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue of P100, P102, P51, P45 .

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Picking of of E. coli XL1 blue transformed with ligation product of F17+F28

Investigator: Andi

Aim of the experiment: Picking of of E. coli XL1 blue transformed with ligation product of F17+F28.

Procedure:

  • Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x) (
  • 3 colonies were picked.
  • These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight.

Quick Change mutagenesis to remove forbidden restriction sites - SDMIII - of P133 (EreB)

Investigator: Louise

Aim of the experiment: Removal of forbidden restiction site from P133 (EreB).

Procedure: Quickchange - PCR

Reaction batch

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid template (P133 - 1:10 dilution) need to get to 50 ng/µl
1 µl 1:10 dilution of O20 (for P133, 10 µM)
1 µl 1:10 dilution of O21 (for P133, 10 µM)
18 µl ddH2O
1 µl dNTP mix 50mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)


PCR cycling parameters - P133

Segment Cycles Temperature Time
1 1 95 °C 30 s
2 19 95 °C 30 s
52 °C 1 min
68 °C 3 min 30 s
  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

Transformation of the Quickchange product into E. coli XL1 blue

Investigator: Louise

Aim of the experiment: Transformation of E. coli XL1 blue with Quickchangeproduct SDM III of P133.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA was added to 100 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Preparative digestion of t35s(F34) with XbaI & PstI, npt(F25) with EcoRI & SpeI, PIF3(F36) with XbaI & AgeI-HF and P10 with XbaI & PstI as well as with EcoRI & SpeI

Investigator: Louise

Aim of the experiment: Preparative digestion of t35s(F34) with XbaI & PstI, npt(F25) with EcoRI & SpeI, PIF3(F36) with XbaI & AgeI-HF and P10 with XbaI & PstI as well as with EcoRI & SpeI.

Procedure:

  • Batch for preparative digestion of t35s(F34) with XbaI and PstI
volume reagent
25 µl Plasmid DNA P61
5 µl CutSmart Buffer
1 µl XbaI (20 U/µl)
1 µl PstI (20 U/µl)
18 µl ddH2O
=50 µl TOTAL
  • Batch for preparative digestion of npt(F35) with EcoRI and SpeI
volume reagent
25 µl Plasmid DNA P61
5 µl CutSmart Buffer
1 µl EcoRI (20 U/µl)
1 µl SpeI (20 U/µl)
18 µl ddH2O
=50 µl TOTAL
  • Batch for preparative digestion of PIF3(F36) with XbaI and AgeI-HF
volume reagent
25 µl Plasmid DNA P61
5 µl CutSmart Buffer
1 µl XbaI (20 U/µl)
1 µl AgeI-HF (20 U/µl)
18 µl ddH2O
=50 µl TOTAL
  • Batch for preparative digestion of P10 with XbaI & PstI for backbone isolation.
volume reagent
20 µl Plasmid DNA P96
4 µl CutSmart Buffer
1 µl XbaI (20 U/µl)
1 µl PstI (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P10 with EcoRI and SpeI for backbone isolation.
volume reagent
20 µl Plasmid DNA P96
4 µl CutSmart Buffer
1 µl EcoRI (20 U/µl)
1 µl SpeI (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Incubation for 2.5 h at 37 °C.
  • The resulting fragments were named: ?????
  • 5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batches and 4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and were loaded on a 1% agarose gel for preparative gelelectrophoresis.
  • Preparative gelelectrophoresis was performed at 90 V for 90 min.

TUM13 20130529 prep Verd F34 Xba.PstI F35 EcoRI.SpeI F36 XbaI.AgeI.png

1 kbp ladder DNA ladder Digestion of PCR product t35s (F34) with XbaI & PstI = F38 Digestion of PCR product npt casette (F25) with EcoRI & SpeI = F39 Digestion of PCR product PIF3 (F36) with XbaI & AgeI-HF = F40
as expected, lower band was cut out as expected, bright band was cut out as expected, bright band was cut out


TUM13 20130529 prep Verdau P10 EcoRI.SpeI P10 XbaI.PstI.png

1 kbp ladder DNA ladder Digestion of P10 with EcoRI & SpeI = F41 Digestion of P10 with XbaI & PstI = F42
as expected, lower band was cut out as expected, lower band was cut out
  • Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.

Transformation of E. coli XL1 blue with pSB1C3 containing a NLS from SV40 (BBa_K801030)

Investigator: Florian, Jeff

Aim of the experiment: Transformation of E. coli XL1 blue with pSB1C3 containing a NLS from SV40 (BBa_K801030).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA was added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

PCR of pActin (P143)

Investigator: Florian, Jeff

Aim of the experiment: PCR of pActin (P143).

Procedure:

Operational sequence:

  • PCR reaction mixture
volume reagent
10 µl 5x OneTaq Reaction Buffer
1 µl 10 mM dNTPs
1 µl 10 µM Forward Primer (P143:O62 (pActin_for))
1 µl 10 µM Reverse Primer (P143:O63 (pActin_rev))
0.25 µL OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl Plasmid DNA (P143)
35.75 µL ddH2O Water
=50 µL TOTAL
  • Mix with pipette
  • The PCR program was performed after following scheme:
Initial denaturation 94 °C 30 s
30 cycles 94 °C 30 s
51 °C 60 s
68 °C 80 s
Final extension 68 °C 5 min
Hold 4 °C infinite
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

Results:

  • After analytical gelelectrophoresis there was no product visible. PCR did't work!

Ligation of F17+F40 (PIF3 in pSB1C3(RFC25)), F42+F38 (t35S in pSB1C3(RFC10)) and F41+F39(npt-casette in pSB1C3(RFC10))

Investigator: Jeff

Aim of the experiment: Ligation of F17+F40 (PIF3 in pSB1C3(RFC25)), F42+F38 (t35S in pSB1C3(RFC10)) and F41+F39(npt-casette in pSB1C3(RFC10)).

Procedure:

  • Ligation batch for F17+F40:
volume reagent
1.63 µl F17 (61.4 ng/µl, 2086 bp)
1.88 µl F40 (22.8 ng/µl, ~297 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
13.49 µl ddH2O
=20 µl TOTAL
  • Ligation batch for F42+F38:
volume reagent
1.36 µl F42 (73.5 ng/µl, 2070 bp)
3.42 µl F38 (9.2 ng/µl, 217 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
12.22 µl ddH2O
=20 µl TOTAL
  • Ligation batch for F41+F39:
volume reagent
1.11 µl F41 (89.9 ng/µl, 2070 bp)
13.74 µl F39 (16.0 ng/µl, 1517 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
2.15 µl ddH2O
=20 µl TOTAL
  • Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
  • Cycled ligation has been performed after following protocol in a thermocycler:
99 cycles 12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
Hold 16 °C infinite
  • Lid temperature = 37 °C

Thursday, May 30th

Gradient PCR of pActin (P143)

Investigator: Ingmar

Aim of the experiment: PCR of pActin

Procedure:

Operational sequence:

  • PCR reaction mixture
volume reagent
10 µl 5x OneTaq Reaction Standard Reaction Buffer
1 µl 10 mM dNTPs
1 µl 10 µM Forward Primer O62 (pActin_for)
1 µl 10 µM Reverse Primer O63 (pActin_rev)
0.25 µL OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl Plasmid DNA P143
35.75 µL ddH2O Water
=50 µL TOTAL
  • PCR reaction mixture
volume reagent
10 µl 5x OneTaq GC Reaction Buffer
1 µl 10 mM dNTPs
1 µl 10 µM Forward Primer O62 (pActin_for)
1 µl 10 µM Reverse Primer O63 (pActin_rev)
0.25 µL OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl Plasmid DNA P143
35.75 µL ddH2O Water
=50 µL TOTAL
  • Mix with pipette
  • The PCR program was performed after following scheme:
Initial denaturation 94 °C 30 s
30 cycles 94 °C 30 s
47 °C 60 s
68 °C 120 s
Final extension 68 °C 5 min
Hold 4 °C infinite

A temperature gradient was applied beginning at 47°C and ranging to 51°C. For each integer (47, 48, 49, 50 and 51°C) a single PCR batch was used.

  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.


Analytical gelelectrophoresis Procedure:

  • 4 µl of each PCR product were mixed seperately with 0.44 µl of DNA loading buffer (10x) and analytical gelelectrophoresis was performed at 1% agarose gel for 60 min at 90 V.

TUM13 20130530 grad PCR P143 O62.O63 GCbuffer standardbuffer.png

1 kbp ladder F45 GC buffer F46 GC buffer F47 GC buffer F47 GC buffer F49 GC buffer normal buffer normal buffer normal buffer normal buffer normal buffer 1 kbp ladder
PCR products has expected length PCR products has expected length PCR products has expected length PCR products has expected length PCR products has expected length PCR products has expected length, some byproducts PCR products has expected length, some byproducts PCR products has expected length, some byproducts PCR products has expected length, some byproducts PCR products has expected length, some byproducts

Miniprep of overnight cultures of transformated E. coli XL1 blue of F17+F28 (FluA in pSB1C3 RFC25)

Investigator: Jeff, Flo

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue of F17+F28 (FluA in pSB1C3 RFC25).

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analytical digestion and gelelectrophoresis of ligation products F17+F28 (FluA in pSB1C3 RFC25)

Investigator: Flo, Jeff

Aim of the experiment: Analytical digestion and gelelectrophoresis of ligation products F17+F28 (FluA in pSB1C3 RFC25).

Procedure:

  • Analytical digestion of ligation products of F17+F28 (FluA in pSB1C3 RFC25) with XbaI & AgeI.
volume reagent
2.5 µl P150/P151/P152/P153
2 µl CutSmart Buffer (10x)
0.25 µl XbaI
0.25 µl AgeI-HF
15 µl ddH2O
=20 µl TOTAL
  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.

TUM13 20130530 anal Verd P150-P153 XbaI.AgeI.png

1 kbp ladder DNA ladder# Digestion of P150 with XbaI&AgeI-HF Digestion of P151 with XbaI&AgeI-HF Digestion of P152 with XbaI&AgeI-HF Digestion of P153 with XbaI&AgeI-HF
as expected as expected as expected as expected

Preparative gelelectrophoresis of oligohybridization products F31 & F32

Investigator: Flo, Jeff

Aim of the experiment: Preparative gelelectrophoresis of oligohybridization products F31 & F32.

Procedure:

  • 50 µl of the 1:10 dilution of F31 and F32 were mixed with 5 µl of DNA loading buffer (10x).
  • Preparative gelelectrophoresis was performed 1.5 h on a 2% agarose gel at 90 V.

TUM13 20130530 F31 F32.png

1 kbp ladder F31 F32
  • Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
  • Gelpurified F31 and F32 were labelled as F43 and F44.

Transformation of E. coli XL1 blue with F17+F40 (PIF3 in pSB1C3(RFC25)), F42+F38 (t35S in pSB1C3(RFC10)) and F41+F39(npt-casette in pSB1C3(RFC10))

Investigator: Florian, Jeff

Aim of the experiment: Transformation of E. coli XL1 blue with F17+F40 (PIF3 in pSB1C3(RFC25)), F42+F38 (t35S in pSB1C3(RFC10)) and F41+F39(npt-casette in pSB1C3(RFC10)).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA were added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on a new chlorampenicol plates.

Ligation of F42+F43 and F17+F32

Investigator: Rosario

Aim of the experiment: Ligation of F42+F43 and F17+F32.

Procedure:

  • Ligation batch for F42+F43:
volume reagent
1.36 µl F42 (73.5 ng/µl, 2070 bp)
1.51 µl F43 (5.4 ng/µl, 34 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
14.13 µl ddH2O
=20 µl TOTAL
  • Ligation batch for F17+F32:
volume reagent
1.63 µl F17 (61.4 ng/µl, 2086 bp)
0.25 µl F32 (2510.8 ng/µl, 49 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
15.12 µl ddH2O
=20 µl TOTAL
  • Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
  • Cycled ligation has been performed after following protocol in a thermocycler:
99 cycles 12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
Hold 16 °C infinite
  • Lid temperature&

Picking of of E. coli XL1 blue transformed with EreB SDMIII (P149) and BBa_K801030 (SV40 NLS)

Investigator: Louise

Aim of the experiment: Picking of of E. coli XL1 blue transformed with EreB SDMIII (P149) and BBa_K801030 (SV40 NLS).

Procedure:

  • Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
  • 5 colonies of EreB SDMIII (P149) and 2 colonies of BBa_K801030 (SV40 NLS) were picked.
  • These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight.

Friday, May 31st

Miniprep of overnight cultures of transformated E. coli XL1 blue with P149 (QCIII of EreB) and BBa_K801030 (SV40 NLS)

Investigator: Louise, Jeff, Katrin

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with P149 (QCIII of EreB) and BBa_K801030 (SV40 NLS)

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analytical digestion and gelelectrophoresis of P149 (QCIII of EreB) with EcoRI-HF and PstI-HF

Investigator: Louise, Jeff, Katrin

Aim of the experiment: Analytical digestion and gelelectrophoresis of P154-P158 (QCIII of EreB) with EcoRI-HF and PstI-HF to check if QCII was successful

Procedure:

  • Analytical digestion of P154-P158 (QCIII of EreB) with EcoRI-HF and PstI-HF
volume reagent
2.5 µl P149-P158/P133 (control before QCIII)
2 µl CutSmart Buffer (10x)
0.25 µl EcoRI-HF
0.25 µl PstI-HF
15 µl ddH2O
=20 µl TOTAL
  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.

TUM13 20130531 anal Verd QCIII P133 P154-158 EcoRI,PstII.png

1 kbp ladder DNA ladder# Digestion of P133 with EcoRI-HF and PstI-HF Digestion of P154 with EcoRI-HF and PstI-HF Digestion of P155 with EcoRI-HF and PstI-HF Digestion of P156 with EcoRI-HF and PstI-HF Digestion of P157 with EcoRI-HF and PstI-HF Digestion of P158 with EcoRI-HF and PstI-HF
as expected as expected as expected as expected as expected as expected

Sequencing of P157 and P160

Investigators: Louise, Jeff, Katrin

Aim of the experiment: Sequencing of QCIII product (EreB) and BBa_K801030 (SV40 NLS)

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label Name Barcode1 Barcode2
P157 Miniprep of transformation of QCIII product of P149 (QC - SDMIII - of EreB to remove forbidden PstI) FR01002289 (VR) FR01002290 (VF2)
P160 Miniprep of transformation of BBa_K801030 (SV40 NLS) FR01002291 (VR) FR01002292 (VF2)

Ligation of F29+F30

Investigator: Katrin

Aim of the experiment: Ligation of F29+F30

Procedure:

  • Ligation batch
volume reagent
4.22 µl F29 (23.7 ng/µl, 2110 bp))
10.48 µl F30 (9,5 ng/µl, about 700 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
2.3 µl ddH2O
=20 µl TOTAL


No negative control was prepared because there was not enough vector backbone left Ligation was performed at 37 °C overnight

Quick Change mutagenesis to remove forbidden restriction sites - SDMIII - of P124 (Laccase)

Investigator: Louise, Katrin

Aim of the experiment: Removal of forbidden restiction site from P124 (Laccase).

Procedure: Quickchange - PCR

Reaction batch

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid template (P124 - 1:10 dilution) need to get between 5 and 50 ng/µl
1 µl 1:10 dilution of O30 (for P124, 10 µM)
1 µl 1:10 dilution of O31 (for P124, 10 µM)
18 µl ddH2O
1 µl dNTP mix 50mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)


PCR cycling parameters - P124

Segment Cycles Temperature Time
1 1 95 °C 30 s
2 19 95 °C 30 s
52 °C 1 min
68 °C 4 min
4 °C hold

After the PCR; 1 µl of Dpn1 was added and the reaction mixture was incubated at 37°C for one hour

Preparative digestion of pActin(F46) with XbaI & PstI

Investigator: Ingmar

Aim of the experiment: Preparative digestion of pActin(F46) in order to ligat this fragment in pSB1C3 later on.

Procedure:

  • Batch for preparative digestion of pActint(F46) with XbaI and PstI
volume reagent
25 µl F46 PCR product of pActin
5 µl CutSmart Buffer
1 µl XbaI (20 U/µl)
1 µl PstI (20 U/µl)
18 µl ddH2O
=50 µl TOTAL
  • Incubation for 3 h at 37 °C.
  • 5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batchesand loaded on a 1% agarose gel for preparative gelelectrophoresis.
  • Preparative gelelectrophoresis was performed at 90 V for 90 min.

TUM13 20130531 prep Verd F46 pActin XbaI+PstI.Tif

  • The band was extracted by QIAquick Gel Extraction Kit, QIAGEN. The resulting fragment was named: F50

Ligation of F42+F50 (pActin in pSB1C3)

Investigator: Ingmar

Aim of the experiment: Ligation of F42+F50 (pActin in pSB1C3(RFC25).

Procedure:

  • Ligation batch
volume reagent
1.37 µl F42
13.13 µl F50
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
2.5 µl ddH2O
=20 µl TOTAL
  • Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
  • Cycled ligation has been performed after following protocol in a thermocycler:
99 cycles 12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
Hold 16 °C infinite
  • Lid temperature = 37 °C

Picking of of E. coli XL1 blue transformed with F42+F38 (t35S), F17+F40 (Pif 3) and F41+F39 (npt)

Investigator: Ingmar

Aim of the experiment: Picking of of E. coli XL1 blue transformed with F42+F38 (t35S), F17+F40 (Pif 3) and F41+F39 (npt).

Procedure:

  • The picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover containing 5 mL of LB-medium + 5 µL chloramphenicol(1000x).
  • 3 colonies of each agar plate were picked.
  • These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight.

Transformation of E. coli XL1 blue with ligation products F42+F43 and F17+F32

Investigator: Louise, Jeff

Aim of the experiment: Transformation of E. coli XL1 blue with ligation products F42+F43 and F17+F32.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA was added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on new chlorampenicol plates.

Saturday, June 1st

Transformation of E. coli XL1 blue with ligation products F29+F30 (GFP in pSB1C3), F42+F50 (actin promoter in pSB1C3), F42+F50 (negative control)

Investigator: Katrin

Aim of the experiment: Transformation of E. coli XL1 blue with ligation products F29+F30 (GFP in pSB1C3), F42+F50 (actin promoter in pSB1C3), F42+F50 (negative control)

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 5 µl of DNA were added to 100 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on a new chlorampenicol plates.

Transformation of E. coli XL1 blue with QCIII product of Laccase (SDMIII of P124)

Investigator: Katrin

Aim of the experiment: Transformation of E. coli XL1 blue with QCIII product of Laccase

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA were added to 100 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on a new chlorampenicol plates.

Miniprep of overnight cultures of transformated E. coli XL1 blue with F39 + F41 (npt-casette in pSC1C3),F42+F38 (t35s in pSB1C3), F17+F40 (PIF3 in pSB1C3)

Investigator: Katrin

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with F39 + F41 (npt-casette in pSC1C3),F42+F38 (t35s in pSB1C3), F17+F40 (PIF3 in pSB1C3)

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analytical digestion and gelelectrophoresis of P161-163 (F39 + F41, npt-casette in pSB1C3), P164-166 (F42+F38, t35s in pSB1C3), P167-169 (F17+F40, PIF3 in pSB1C3)

Investigator: Katrin

Aim of the experiment: Analytical digestion and gelelectrophoresis of P161-163 (F39 + F41, npt-casette in pSC1C3) with EcoRI-HF and SpeI-HF, P164-166 (F42+F38, t35s in pSB1C3) with EcoRI-HF and SpeI-HF and P167-169 (F17+F40, PIF3 in pSB1C3) with AgeI-HF and XbaI

Procedure:

  • Analytical digestion of P161-163 (F39 + F41, npt-casette in pSC1C3), P164-166 (F42+F38, t35s in pSB1C3) with EcoRI-HF and SpeI-HF
volume reagent
2.5 µl P161-P166
2 µl CutSmart Buffer (10x)
0.25 µl EcoRI-HF
0.25 µl SpeI-HF
15 µl ddH2O
=20 µl TOTAL
  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.


  • Analytical digestion of P167-169 (F17+F40, PIF3 in pSB1C3) with AgeI-HF and XbaI
volume reagent
2.5 µl P167-P169
2 µl CutSmart Buffer (10x)
0.25 µl AgeI-HF
0.25 µl XbaI
15 µl ddH2O
=20 µl TOTAL
  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.

TUM 13 20130601 anal Verd F41+39 EcoRI SpeI F42+38 EcoRI SpeI F17+40 XbaI+AgeI.png

1 kbp ladder DNA ladder# Digestion of P161 with EcoRI-HF and SpeI-HF Digestion of P162 with EcoRI-HF and SpeI-HF Digestion of P163 with EcoRI-HF and SpeI-HF Digestion of P164 with EcoRI-HF and SpeI-HF Digestion of P165 with EcoRI-HF and SpeI-HF Digestion of P166 with EcoRI-HF and SpeI-HF Digestion of P167 with AgeI-HF and XbaI Digestion of P168 with AgeI-HF and XbaI Digestion of P169 with AgeI-HF and XbaI
as expected as expected as expected as expected as expected as expected as expected as expected as expected

Sunday, June 2nd

Picking of of E. coli XL1 blue transformed with QCIII product of P124 and the ligation products of F17+F44 and F42+F32

Investigator: Andi

Aim of the experiment: Picking of of E. coli XL1 blue transformed with QCIII product of P124 and the ligation products of F17+F32 and F42+F32.

Procedure:

  • The picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover containing 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
  • 4 colonies of each agar plate were picked.
  • These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight.

Week 7

Monday, June 3rd

Analytical digestion and gelelectrophoresis of P170-P173 (F17+F32, SpyTag in pSB1C3 RFC25), P174-P177 (F42+F43, miniMCSII in pSB1C3 RFC10), P178-181 (QCIII von P142, Laccase in pSB1C3 RFC10)

Investigator: Andi, Johanna, Jeff

Aim of the experiment: Analytical digestion and gelelectrophoresis of P170-P173 (F17+F32, SpyTag in pSB1C3 RFC25), P174-P177 (F42+F43, miniMCSII in pSB1C3 RFC10), P178-181 (QCIII von P142, Laccase in pSB1C3 RFC10).

Procedure:

  • Mastermix for analytical digestion of P170-P173 (F17+F32, SpyTag in pSB1C3 RFC25), P174-P177 (F42+F43, miniMCSII in pSB1C3 RFC10) with XbaI+PstI.
volume reagent
18 µl CutSmart Buffer (10x)
2.25 µl EcoRI-HF
2.25 µl SpeI-HF
135 µl ddH2O
=157.5 µl TOTAL
  • 17.5 µl of the mastermix was added to 2.5 µl of plasmid DNA (P170-P177)
  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.


  • Analytical digestion of P178-181 (QCIII von P142, Laccase in pSB1C3 RFC10):
volume reagent
2.5 µl P178-P181
2 µl CutSmart Buffer (10x)
0.25 µl NgoMIV
0.25 µl PstI-HF
15 µl ddH2O
=20 µl TOTAL
  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.

TUM13 20130603 anal Verd P170-P177 EcoRI.PstI.png

1 kbp ladder DNA ladder Digestion of P170 with EcoRI-HF and PstI-HF Digestion of P162 with EcoRI-HF and PstI-HF Digestion of P163 with EcoRI-HF and PstI-HF Digestion of P164 with EcoRI-HF and PstI-HF Digestion of P164 with EcoRI-HF and PstI-HF Digestion of P164 with EcoRI-HF and PstI-HF Digestion of P164 with EcoRI-HF and PstI-HF Digestion of P164 with EcoRI-HF and PstI-HF Digestion of P164 with EcoRI-HF and PstI-HF
as expected, insert too small to be visible as expected, insert too small to be visible as expected, insert too small to be visible as expected, insert too small to be visible as expected, insert too small to be visible as expected, insert too small to be visible as expected, insert too small to be visible as expected, insert too small to be visible as expected, insert too small to be visible


TUM13 20130603 anal Verd P142 NgoMIV.PstI P178-P181 NgoMIV.PstI.png

1 kbp ladder DNA ladder Digestion of P142 with NgoMIV and PstI-HF Digestion of P178 with NgoMIV and PstI-HF Digestion of P179 with NgoMIV and PstI-HF Digestion of P180 with NgoMIV and PstI-HF Digestion of P181 with NgoMIV and PstI-HF
Ctrl (before QuickChange III) as expected, plasmid is only linearized, all NgoMIV sites are removed as expected, plasmid is only linearized, all NgoMIV sites are removed as expected, plasmid is only linearized, all NgoMIV sites are removed as expected, plasmid is only linearized, all NgoMIV sites are removed

Tuesday, June 4th

Sequencing of P162(npt-cassete), P165(t35s), P168(PIF3), P179(LaccQCIII), P177(MiniMCS), P170(Spytag)

Investigators: Louise, Johanna

Aim of the experiment: Sequencing of P162(npt-cassete), P165(t35s), P168(PIF3), P179(LaccQCIII), P177(MiniMCS), P170(Spytag).

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label Name Barcode1 Barcode2
P162 Miniprep of ligation product F39 + F41 (npt-casette in pSB1C3) FR01002331 (VR) FR01002330 (VF2)
P165 Miniprep of ligation product F42+F38 (t35s in pSB1C3) FR01002332 (VF2)
P168 Miniprep of ligation product F17+F40 (PIF3 in pSB1C3) FR01002333 (VF2)
P179 Miniprep of ligation product Laccase QCIII (P142) FR01002335 (VR) FR01002334 (VF2)
P177 Miniprep of ligation product MiniMCSII F42+F43 FR01002336 (VF2)
P170 Miniprep of ligation product F17+F32 (SpyTag) FR01002328 (VF2)

Preparative digestion and gelelectrophoresis of P39(middle-linker) with PstI and AgeI-HF, P20(GFP-Psb1C3) with NgoMIV and PstI, F45(pActin) with XbaI and PstI

Investigator: Louise, Johanna

Aim of the experiment: Preparative digestion of P39(middle-linker) with PstI and AgeI-HF, P20(GFP-Psb1C3) with NgoMIV and PstI, F45(pActin) with XbaI and PstI.

Procedure:

  • Batch for preparative digestion of P39(middle-linker) with PstI and AgeI-HF
volume reagent
15 µl Plasmid DNA P39
4 µl CutSmart Buffer
1 µl AgeI-HF(20 U/µl)
1 µl PstI (20 U/µl)
19 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P20(GFP-Psb1C3) with NgoMIV and PstI
volume reagent
20 µl Plasmid DNA P20
4 µl CutSmart Buffer
1 µl NgoMIV (20 U/µl)
1 µl PstI (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of F45(pActin) with XbaI and PstI
volume reagent
25 µl Fragment DNA F45
5 µl CutSmart Buffer
1 µl XbaI (20 U/µl)
1 µl PstI (20 U/µl)
18 µl ddH2O
=50 µl TOTAL
  • Incubation for 3 h at 37 °C.
  • 5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batches and 4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and were loaded on a 1% agarose gel for preparative gelelectrophoresis.
  • Preparative gelelectrophoresis was performed at 90 V for 90 min.

TUM13 20130604 prep Verd P20 NgoMIV.PstI P39 AgeI.Pst F45 XbaI.PstI.png

1 kbp DNA ladder Digestion of P20 with NgoMIV & PstI = F53 Digestion of P39 with AgeI & PstI = F52 Digestion of F45 with XbaI & PstI = F51
as expected, lower band was cut out as expected, band was cut out as expected, lower band was cut out
  • Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.

Quick Change mutagenesis to remove forbidden restriction sites - SDMIV - of P179 (Laccase)

Investigator: Andi, Jeff

Aim of the experiment: Removal of forbidden restiction site SDM IV from P179 (Laccase).

Procedure: Quickchange - PCR

Reaction batch

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid template (P179 - 1:6 dilution) need to get between 5 and 50 ng/µl
1 µl 1:10 dilution of O32 (for P179, 10 µM)
1 µl 1:10 dilution of O33 (for P179, 10 µM)
18 µl ddH2O
1 µl dNTP mix 50mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)


PCR cycling parameters - P179

Segment Cycles Temperature Time
1 1 95 °C 30 s
2 19 95 °C 30 s
52 °C 1 min
68 °C 4 min
4 °C hold

After the PCR; 1 µl of Dpn1 was added and the reaction mixture was incubated at 37°C for one hour

Preparative digestion and gelelectrophoresis of P182 (Gensynthesis 1) with XbaI and AgeI-HF, P183 (Gensynthesis 2) with XbaI and AgeI-HF

Investigator: Andi, Jeff

Aim of the experiment: Preparative digestion and gelelectrophoresis of P182 (Gensynthese 1) with XbaI and AgeI-HF, P183 (Gensynthese 2) with XbaI and AgeI-HF

Procedure:

  • Batch for preparative digestion of P182 with XbaI and AgeI-HF
volume reagent
15 µl Plasmid DNA P182
4 µl CutSmart Buffer
1 µl AgeI-HF(20 U/µl)
1 µl XbaI (20 U/µl)
19 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P183 with XbaI and AgeI- HF
volume reagent
15 µl Plasmid DNA P183
4 µl CutSmart Buffer
1 µl AgeI-HF (20 U/µl)
1 µl XbaI (20 U/µl)
19 µl ddH2O
=40 µl TOTAL
  • Incubation for 2.5 h at 37 °C.
  • 4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and were loaded on a 1.5% agarose gel for preparative gelelectrophoresis.
  • Preparative gelelectrophoresis was performed at 70 V for 60 min.

TUM13 20130604 prep verdau P182-183 XbaI,AgeI.png

1 kbp DNA ladder Digestion of Gensynthesis 1 with XbaI & AgeI 100 bp DNA ladder Digestion of Gensynthesis 2 with XbaI & AgeI 1 kbp DNA ladder
as expected, fragments (78 bp and 530 bp) were cut out as expected, fragments (111 bp, 206 bp and 359 bp) were cut out
  • Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.

Preparative digestion and gelelectrophoresis of P9 with XbaI and AgeI-HF

Investigator: Andi, Jeff

Aim of the experiment: Preparative digestion and gelelectrophoresis of P9 with XbaI and AgeI-HF

Procedure:

  • Batch for preparative digestion of P9 with XbaI and AgeI-HF
volume reagent
15 µl Plasmid DNA P9
4 µl CutSmart Buffer
1 µl AgeI-HF(20 U/µl)
1 µl XbaI (20 U/µl)
19 µl ddH2O
=40 µl TOTAL
  • Incubation for 2.5 h at 37 °C.
  • The resulting fragments were named: ?????
  • 4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and were loaded on a 1% agarose gel for preparative gelelectrophoresis.
  • Preparative gelelectrophoresis was performed at 70 V for 60 min.

TUM13 20130604 prep verdau P9 XbaI.AgeI.png

  • Gelfragmentation was like expected, both bands were cut out (upper band = PhyB, lower band = pSB1C3 RFC25)
  • Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.

Transformation of E. coli XL1 blue with Quickchange product SDM IV of P179 (Laccase)

Investigator: Andi, Jeff

Aim of the experiment: Transformation of E. coli XL1 blue with Quickchange product SDM IV of P179 (Laccase).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 2x 10 µl of DNA was added seperately into 2x 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on new chlorampenicol plates.

Transformation of E. coli XL1 blue with P39

Investigator: Louise, Johanna

Aim of the experiment: Transformation of E. coli XL1 blue with P39

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA was added seperately into 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on new chlorampenicol plates.

Preparative digestion and gelelectrophoresis of P39 with AgeI & PstI, P20 with NgoMIV & PstI

Where is the snapshot of the gel =DDDD

Investigator: Johanna, Louise

Aim of the experiment:Preparative digestion of P39 with AgeI & PstI, P20 with NgoMIV & PstI to fusion Middle linker of P39 with GFP of P20.

Procedure:

Batch for preparative digestion of P39 (Middle-Linker) with PstI and AgeI.

volume reagent
20 µl P39
4 µl NEBuffer 4
0.4 µl BSA (100x)
2 µl AgeI-HF
2 µl PstI
11.6 µl ddH2O
=40 µl TOTAL

Batch for preparative digestion of P20 (GFP-Psb1C3) with NgoMIV and PstI.

volume reagent
20 µl P45
4 µl NEBuffer 4
0.4 µl BSA (100x)
2 µl NgoMIV
2 µl PstI
11.6 µl ddH2O
=40 µl TOTAL
  • Incubation for 150 min at 37 °C.
  • Preparative gelelectrophoresis was performed at 90 V for 1 h.

File:FOTOFOTO.png

1 kbp ladder P20 digested with NgoMIV&PstI (=F30) P39 digested with NgoMIV&PstI (=F30)
lower band was cut out band was cut out
  • Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.

Ligation of F58+F59, F55+F59, F54+F59, F56+F59, F57+F59, F52+F53, F42+F51

Investigator: Jeff, Andi, Johanna, Louise

Aim of the experiment: Ligation of F53+F59, F58+F59, F55+F59, F54+F59, F56+F59, F57+F59, F52+F53, F42+F51.

Procedure:

  • Ligation batch for F54+F59 :
volume reagent
2.2 µl F54 (5.9 ng/µl, 90 bp)
1.5 µl F59 (66.7 ng/µl, 2100 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
13.3 µl ddH2O
=20 µl TOTAL
  • Ligation batch for F55+F59:
volume reagent
11.6 µl F55 (6.4 ng/µl, 520 bp)
1.5 µl F59 (66.7 ng/µl, 2100 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
3.9 µl ddH2O
=20 µl TOTAL
  • Ligation batch for F56+F59:
volume reagent
3.3 µl F56 (4.7 ng/µl, 110 bp)
1.5 µl F59 (66.7 ng/µl, 2100 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
12.2 µl ddH2O
=20 µl TOTAL
  • Ligation batch for F57+F59:
volume reagent
3.1 µl F57 (9.1 ng/µl, 200 bp)
1.5 µl F59 (66.7 ng/µl, 2100 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
12.4 µl ddH2O
=20 µl TOTAL
  • Ligation batch for F58+F59:
volume reagent
4.4 µl F58 (12.9 ng/µl, 400 bp)
1.5 µl F59 (66.7 ng/µl, 2100 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
11.1 µl ddH2O
=20 µl TOTAL
  • Negative Control Ligation batch for F59:
volume reagent
1.5 µl F59 (66.7 ng/µl, 2100 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
15.5 µl ddH2O
=20 µl TOTAL
  • Ligation batch for F52+F53:
volume reagent
10 µl F52 (9.9 ng/µl, XXX bp)
7 µl F53 (12.2 ng/µl, XXX bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F42+F51:
volume reagent
1.36 µl F42 (XXX ng/µl, XXX bp)
15.64 µl F51 (8.2 ng/µl, 1237 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
  • Cycled ligation has been performed after following protocol in a thermocycler:
99 cycles 12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
12 °C 60 s
22 °C 60 s
Hold 16 °C infinite
  • Lid temperature = 37 °C

Plating of received E. coli containing biobricks

Investigator: Jeff, Andreas

Aim of the experiment: The received biobricks were already transformed in E. coli and were in an agar stabs. These E. coli cells were transferred with an inoculation loop on antibiotic selection plates and were incubated over night.

Operational sequence:

  • Bacterias containing plasmids with biobricks were transferred with a sterile inoculation loop on antibiotic plates and were incubated at 37 °C overnight.
  • The biobricks were:
Name Function
BBa_J61032 Alkaline Phosphatase
BBa_K426020 Self lysis device
BBa_K729004 Nuclease from S. Aureus
BBa_K365001 PIF6
BBa_K648011 RBS B0034 with cI repressor C0051 under the control of constitutive promoter J23113

Wednesday, June 5th

Transformation of E. coli XL1 blue transformed with F59+F54, F59+F55, F59+F56, F59+F57, F59+F58, F42+F51 and F52+F53

Investigator: Florian, Rosario, Louise, Jeff

Aim of the experiment: Transformation of E. coli XL1 blue transformed with F59+F54, F59+F55, F59+F56, F59+F57, F59+F58, F42+F51 and F52+F53.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA was added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on plates containing antibiotics.

Picking of of E. coli XL1 blue transformed with BBa_K365001 (PIF6), BBa_K729004 (Nuclease from Staphylococcus aureus), BBa_K648011 (XylE RFC25), BBa_J61032 (Alkaline phosphotase), BBa_K426020 (BBa_K426020)

Investigator: Rosario, Florian, Louise, Jeff

Aim of the experiment: Picking of of E. coli XL1 blue transformed with BBa_K365001 (PIF6), BBa_K729004 (Nuclease from Staphylococcus aureus), BBa_K648011 (XylE RFC25), BBa_J61032 (Alkaline phosphotase), BBa_K426020 (BBa_K426020).

Procedure:

  • Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x) or ampicillin(1000x).
  • 2 colonies of each plate were picked.
  • These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight.

Thursday, June 6th

Miniprep of overnight cultures of transformated E. coli XL1 blue with P39, P20, genesynthesis 1&2 and BBa_J61032, BBa_K426020, BBa_K729004, BBa_K365001, BBa_K648011

Investigator: Ingmar

Aim of the experiment:Miniprep of overnight cultures of transformated E. coli XL1 blue with P39, P20, genesynthesis 1&2 and BBa_J61032, BBa_K426020, BBa_K729004, BBa_K365001, BBa_K648011

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
  • The resulting DNA were eluted in tubes labeled as follows:
Content New Tube
P39P184
P39P185
P20P186
P20P187
BBa_J61032P188
BBa_J61032P189
BBa_K426020P190
BBa_K426020P191
BBa_K729004P192
BBa_K729004P193
BBa_K365001P194
BBa_K365001P195
BBa_K648011P196
BBa_K648011P197
Genesynthesis 2P198
Genesynthesis 2P199
Genesynthesis 1P200
Genesynthesis 1P201

Analytical digestion and gelelectrophoresis of P188-P197 (latest received biobricks: BBa_J61032, BBa_K426020, BBa_K729004, BBa_K365001, BBa_K648011)

Investigator: Jeff

Aim of the experiment: Analytical digestion and gelelectrophoresis of P188-P197 (latest received biobricks: BBa_J61032, BBa_K426020, BBa_K729004, BBa_K365001, BBa_K648011).

Procedure:

volume reagent
2 µl CutSmart Buffer (10x)
0.25 µl EcoRI-HF
0.25 µl SpeI-HF
15 µl ddH2O
2.5 µl Plasmid
=20 µl TOTAL

Mastermix (12x)

volume reagent
24 µl CutSmart Buffer (10x)
3 µl EcoRI-HF
03 µl SpeI-HF
180 µl ddH2O
=210 µl TOTAL
  • 17.5 µl Mastermix + 2.5 µl Plasmid each
  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V


TUM13 20130606 anal Verd P188-P197 EcoRI.SpeI.png

1 kb Marker P188 Alk.Phos. P189 Alk.Phos. P190 self lysis device P191 self lysis device P192 Nuclease P193 Nuclease P194 PIF6 P195 PIF6 P196 RBS P197 RBS
successful successful successful successful successful successful successful successful successful successful

Picking of of E. coli XL1 blue transformed with the ligation products of F52+F53, F59+F58, F59+F54, F59+F55, F59+F57 and F59+F56

Investigator: Ingmar

Aim of the experiment: Picking of of E. coli XL1 blue transformed with the ligation products of F52+F53, F59+F58, F59+F54, F59+F55, F59+F57 and F59+F56

Procedure:

  • The picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover containing 4 mL of LB-medium + 4 µL chloramphenicol(1000x) or ampecillin (1000x).
  • 2 colonies of each agar plate were picked.
  • These tubes were transferred in a cell culture shaker at 37 °C and were incubated overday.
  • The ligation of F42 and F51 was not successful and has to be repeated. One reason for the failure could be secondary structures of the promoter. Therefore the new ligation batch should be heated to 95°C for a while before adding the T4 ligase.

PCR of P192 (Thermonuclease, O64 & O65) and P194 (PIF6, O42 & O43)

Investigator: Jeff

Aim of the experiment: PCR of P192 (Thermonuclease, O64 & O65) and P194 (PIF6, O42 & O43).

Procedure:

Operational sequence:

  • PCR reaction mixture
volume reagent
10 µl 5x OneTaq Standard Reaction Buffer
1 µl 10 mM dNTPs
1 µl 10 µM Forward Primer (P192: O64 (NucA_fw); P194: O42 (PIF6_fw))
1 µl 10 µM Reverse Primer (P192: O65 (NucA_rv); P194: O43 (PIF6_rv))
0.25 µL OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl 1:1000 dilution of plasmid DNA (P192; P194)
35.75 µL ddH2O Water
=50 µL TOTAL
  • Mix with pipette
  • The gradient PCR program was performed after following scheme with following conditions:
Initial denaturation 94 °C 30 s
30 cycles 94 °C 30 s
55 °C 60 s
68 °C 30 s
Final extension 68 °C 5 min
Hold 4 °C infinite
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
  • PCR product of P192 = F61; PCR product of 194 = F62

QuikChange mutagenesis to remove forbidden restriction sites - SDMIV - of P179 (Laccase)

Investigator: Jeff

Aim of the experiment: Removal of forbidden restiction site from P179 (Laccase).

Procedure: Quickchange - PCR

Reaction batch

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
0.5 µl Plasmid template (P179 - 1:10 dilution) need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl 1:10 dilution of O32 (10 µM)
1 µl 1:10 dilution of O33 (10 µM)
18.5 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)


PCR cycling parameters - P179

Segment Cycles Temperature Time
1 1 95 °C 30 s
2 19 95 °C 30 s
52 °C 1 min
68 °C 4 min
4 °C hold

After the PCR; 1 µl of Dpn1 was added and the reaction mixture was incubated at 37°C for one hour

Analytical gelelectrophoresis of F61 (PCR product of P192, Thermonuclease), F62 (PCR product of P194, PIF6) and the DpnI digestion of the SDMIV product of P179

Investigator: Rosario, Andi, Jeff

Aim of the experiment: Analytical gelelectrophoresis of F61 (PCR product of P192, Thermonuclease), F62 (PCR product of P194, PIF6) and the DpnI digestion of the SDMIV product of P179.

Procedure:

  • 4 µl of F61 or F62 or the DpnI digestion of the SDMIV product of P179 were mixed with 0.444 µl of DNA loading buffer (10x)
  • Analytical gelelectrophoresis was performed at 90 V for 1 h on a 1% agarose gel.

TUM13 20130606 PCR F61 F62 QCIV- P179.png

1 kbp DNA ladder PCR product F61 PCR product F62 DpnI digestion of QCIV of P179
successful successful successful

Ligation of F42+F51

Investigator: Ingmar

Aim of the experiment: Ligation of F42+F51.

Procedure:

  • Ligation batch for F42+F51:
volume reagent
0.68 µl F42 (73.5 ng/µl, 2070 bp)
15.64 µl F51 (10.93 ng/µl, 1237 bp)
5.39 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
  • The reaction batch was heated up to 95°C for 5 min before the enzyme and buffer were added.
  • The ligation was performed for two hours at room temperature.
  • Lid temperature = 37 °C

Transformation of E. coli XL1 blue with F42+F51 (pActin in pSB1C3 RFC10) and DpnI digestion of the SDMIV product of P179 (Laccase)

Investigator: Katrin

Aim of the experiment: Transformation of E. coli XL1 blue with F42+F51 (pActin in pSB1C3 RFC10 )and DpnI digestion of the SDMIV product of P179 (Laccase).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA was added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on plates containing antibiotics (CamR).

Sequencing of Biobricks P188/189 (alk.Phos., 1475 bp), P190/191 (self lysis device, 2910 bp), P192/193 (Nuclease, 561 bp), P194/195 (PIF6, 300 bp), P196/197 (RBS, 918 bp)

Investigators: Johanna

Aim of the experiment: Sequencing of the Biobrick MiniPreps

Procedure: Peparation of DNA for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng/µl) and 2 µl sequencing primer 10 µM):


Label Konz. ng/µl Vol. BB µl Vol H2O µl
P188 160.6 5 10
P189 123.6 7.5 7.5
P190 319.2 3 12
P191 127.0 10 5
P192 336.9 3 12
P193 230.6 4 11
P194 156.6 5 10
P195 197.4 4 11
P196 90.3 10 5
P197 230.2 4 11


The different genes received the following barcodes:

Label Name Barcode1 Barcode2
P188 alk.Phos. FR01002327 (O3) FR01002326 (O4)
P189 alk.Phos. FR01002325 (O3) FR01002320 (O4)
P190 self lysis device FR01002319 (O3) FR01002318 (O4)
P191 self lysis device FR01002317 (O3) FR01002316 (O4)
P192 Nuclease FR01002315 (O3) - (O4)
P193 Nuclease FR01002314 (O3) - (O4)
P194 PIF6 FR01002313 (O3) - (O4)
P195 PIF6 FR01002312 (O3) - (O4)
P196 RBS FR01002324 (O3) FR01002323 (O4)
P197 RBS FR01002322 (O3) FR01002321 (O4)

Preparative digestion and gelelectrophoresis of F61 (Thermonuclease), F62 (PIF6) , P165 (t35S), F31 (miniMCSII)

Investigator: Rosario, Andi, Florian, Johanna, Jeff

Aim of the experiment: Preparative digestion and gelelectrophoresis of F61 (Thermonuclease), F62 (PIF6) , P165 (t35S), F31 (miniMCSII.

Procedure:

  • Batch for preparative digestion of F61 (Thermonuclease) with XbaI & AgeI:
volume reagent
25 µl PCR product F61
5 µl CutSmart Buffer
1 µl XbaI(20 U/µl)
1 µl AgeI-HF (20 U/µl)
18 µl ddH2O
=50 µl TOTAL
  • Batch for preparative digestion of F62 (PIF6) with XbaI & AgeI:
volume reagent
25 µl PCR product F62
5 µl CutSmart Buffer
1 µl XbaI(20 U/µl)
1 µl AgeI-HF (20 U/µl)
18 µl ddH2O
=50 µl TOTAL
  • Batch for preparative digestion of P165 (t35S) with XbaI:
volume reagent
20 µl Plasmid DNA P165
4 µl CutSmart Buffer
1 µl XbaI (20 U/µl)
15 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of F31 (miniMCSII):
volume reagent
10 µl Oligohybridization F31
5 µl CutSmart Buffer
1 µl SpeI-HF (20 U/µl)
34 µl ddH2O
=40 µl TOTAL
  • Incubation for 3 h at 37 °C.
  • 5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batches and 4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and were loaded on an 2% agarose gel for preparative gelelectrophoresis (1% agarose gel for digestion of P165).
  • Preparative gelelectrophoresis was performed at 90 V for 60 min.

TUM13 20130606 prep Verd F31 SpeI F61 XbaI.AgeI F62 XbaI.AgeI.png

1 kbp DNA ladder Digestion of F31 with SpeI-HF Digestion of F61 with XbaI & AgeI-HF Digestion of F62 with XbaI & AgeI-HF
Fragment length as expected; band was cut out Fragment length as expected; band was cut out Fragment length as expected; band was cut out


TUM13 20130606 prep Verd P165 XbaI.png


1 kbp DNA ladder Digestion of P165 with XbaI
Fragment length as expected; band was cut out
  • Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.

Dephosphorylation of preparative digestion of P165 with FastAP

Investigator: Rosario, Jeff

Aim of the experiment: Dephosphorylation of preparative digestion of P165 with FastAP to prevent re-ligation.

Procedure:

  • After preparative digestion, gelelectrophoresis and gelextraction of P165, the digestion product was purified with QIAquick PCR Purification Kit, Qiagen.
  • Batch for the dephosphorylation of digestion product of P165
volume reagent
30 µl Digestion product of P165
4 µl 10X Fast AP buffer
2 µl FastAP Thermosensitive Alkaline Phosphatase
4 µl ddH2O
=40 µl TOTAL
  • The reaction batch was spun briefly and incubated at 37 °C for 10 min.
  • The reaction was stopped by heating to 75 °C for 5 min.
  • The dephosphorylized digestion product of P165 product was purified with QIAquick PCR Purification Kit, Qiagen.
  • The resulting product was labelled as F63

Ligation of F58+F59, F55+F59, F54+F59, F56+F59, F57+F59, F52+F53, F42+F51

Investigator: Jeff, Rosario

Aim of the experiment: Ligation of F53+F59, F58+F59, F55+F59, F54+F59, F56+F59, F57+F59, F52+F53, F42+F51.

Procedure:

  • Ligation batch for F59+F65 (Thermonuclease in pSB1C3 RFC25):
volume reagent
1.5 µl F59 (66.7 ng/µl, 2086 bp)
4.56 µl F65 (14.1 ng/µl, 447 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
10.94 µl ddH2O
=20 µl TOTAL
  • Ligation batch for F59+F66:
volume reagent
1.5 µl F59 (66.7 ng/µl, 2086 bp)
3.78 µl F66 (11.3 ng/µl, 297 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
11.72 µl ddH2O
=20 µl TOTAL
  • Ligation batch for F63+F64:
volume reagent
12.05 µl F63 (8.3 ng/µl, 2287 bp)
4.95 µl F64 (4.3 ng/µl, 22 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
  • Ligation was performed at 16 °C overnight.

Friday, June 7th

Picking of QC IV Laccase

Investigator: Johanna

Aim of the study: Picking of QC IV Laccase (concentrated) and QC IV Laccase (diluted)

Operational sequence:

  • Picking and overnight culture after standard laboratory's protocol. (4 µl CamR and 4 ml LB-medium)
  • 3 colonies were picked for every Transformation product.

Preparation of Knop-Medium for Moss

Investigator: Andi

Aim of the experiment: Preparation of Knop-Medium for Moss

Procedure:

  • All four prepared stocks have been: 25g/L KH2PO4; 25g/L KCl; 25g/L MgSO4 x 7 H2O; 100g/L Ca(NO3)2
  • Volume of prepared Medium: 2.6L
  • 10 ml of each stock were converted into a 5L Erlenmeyer flask and filled up to 2.6L with destilled water (ELGA); 32.5mg FeSO4 x 7 H2O were added; the pH was set to 5.8 with KOH/NaOH
  • 200ml of the prepared medium was filled into three 500ml Erlenmeyer flasks, so each Erlenmeyer flask contained 200 ml of the Knop-Medium; the rest (2L) was left in the 5L EM flask
  • All flasks have been autoclaved

Miniprep of overnight cultures of transformated E. coli XL1 blue with F52+F53, F59+F54, F59+F55, F59+F56, F59+F57, F59+F58

Investigator: Rosario

Aim of the experiment:Miniprep of overnight cultures of transformated E. coli XL1 blue with F52+F53, F59+F54, F59+F55, F59+F56, F59+F57, F59+F58

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
  • The resulting DNA were eluted in tubes labeled as follows:
Content New Tube
F52+F53 IP202
F52+F53 II203
F59+F54 IP204
F59+F54 IIP205
F59+F55 IP206
F59+F55 IIP207
F59+F56 IP208
F59+F56 IIP209
F59+F57 IP210
F59+F57 IIP211
F59+F58 IP212
F59+F58 IIP213


Transformation of E. coli XL1 blue with F59 + F65 (Thermonuclease in pSB1C3), F59 + F66 (PIF6 in pSB1C3) and F63 + F64 (MiniMCS + t35S in pSB1C3)

Investigator: Florian

Aim of the experiment: Transformation of E. coli XL1 blue with F59 + F65 (Thermonuclease in pSB1C3), F59 + F66 (PIF6 in pSB1C3) and F63 + F64 (MiniMCS + t35S in pSB1C3).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of ligation product were added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of each tube were plated on agarose plates containing antibiotics (CamR).
  • The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on plates containing antibiotics (CamR).

Sequencing of P204 (Ligation Product F59+F54), P206 (Ligation Product F59+F55), P208 (Ligation Product F59+F56), P210 (Ligation Product F59+F57), P212 (Ligation Product F59+F58)

A monster is born!

Investigators: Louise

Aim of the experiment: Sequencing of Ig-Kappa (P204), Nanoluciferase (P206), SigP (P208), Transmembrand. (P210) and SpyCatcher (P212) Ligations in pSB1C3 RFC25

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label Name Barcode (O3)
P204 Ig-Kappa-SigP + pSB1C3 RFC25 FR01002304
P206 Nanoluciferase + pSB1C3 RFC25 FR01002303
P208 SERK-SigP + pSB1C3 RFC25 FR01002302
P210 Transmembrand + pSB1C3 RFC25 FR01002301
P212 SpyCatcher + pSB1C3 RFC25 FR01002300

Analytical digestion and gelelectrophoresis of P202-P213

Investigator: Katrin

Aim of the experiment: Analytical digestion and gelelectrophoresis of P202-P213

Procedure:

  • Mastermix for analytical digestion of P202-P213 with XbaI+AgeI
volume reagent
30 µl CutSmart Buffer (10x)
3.75 µl XbaI
3.75 µl SpeI-HF
225 µl ddH2O
=262.5 µl TOTAL
  • 17.5 µl of the mastermix was added to 2.5 µl of plasmid DNA (P202-P203)
  • Incubation for 90 min at 37 °C.
  • Inaddition, P203 was digested for 4 and for 8 seconds in the microwave, a negative control was incubated at room temperature
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.


TUM13 20130607 anal Verd P202-P210 XbaI.AgeI.png


1 kbp ladder DNA ladder Digestion of P202 with XbaI und AgeI (GFP + linker) Digestion of P203 with XbaI und AgeI (GFP + linker) Digestion of P204 with XbaI und AgeI Digestion of P205 with XbaI und AgeI Digestion of P206 with XbaI und AgeI Digestion of P207 with XbaI und AgeI Digestion of P208 with XbaI und AgeI Digestion of P209 with XbaI und AgeI Digestion of P210 with XbaI und AgeI
as expected as expected not as expected not as expected not as expected not as expected not as expected not as expected as expected (SERK-TMD)


TUM13 20130607 anal Verd P211-P213 XbaI.AgeI Katrinspielt P203 XbaI.AgeI.png

1 kbp ladder DNA ladder Digestion of P211 with XbaI und AgeI Digestion of P212 with XbaI und AgeI Digestion of P213 with XbaI und AgeI Digestion of P203 with XbaI und AgeI (microwave 4 seconds) Digestion of P203 with XbaI und AgeI (microwave 8 seconds) Digestion of P203 with XbaI und AgeI (control)
as expected (SERK-TMD) not as expected as expected interesting interesting interesting

Saturday, June 8th

Miniprep of overnight cultures of transformated E. coli XL1 blue QC IV Laccase

Investigator: Rosario

Aim of the experiment:Miniprep of overnight cultures of transformated E. coli XL1 blue QC IV Laccase

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
  • The resulting DNA were eluted in tubes labeled as follows:
Content New Tube
QC IV Laccase clone 1P214
QC IV Laccase clone 2P215
QC IV Laccase clone 3P216
QC IV Laccase clone 4P217
QC IV Laccase clone 5P218
QC IV Laccase clone 6P219

Picking of F59 + F65 (Thermonuclease in pSB1C3), F59 + F66 (PIF6 in pSB1C3) and F63 + F64 (MiniMCS + t35S in pSB1C3)

Investigator: Rosario

Aim of the study: Picking of F59 + F65 (Thermonuclease in pSB1C3), F59 + F66 (PIF6 in pSB1C3) and F63 + F64 (MiniMCS + t35S in pSB1C3) Operational sequence:

  • Picking and overnight culture after standard laboratory's protocol. (4 µl CamR and 4 ml LB-medium)
  • 2 colonies were picked for every Transformation product.


Preparative digestion and gelelectrophoresis of F47 (pActin, fragment named F68) with XbaI/SpeI, P202 (GFP+linker, fragment named F69) with NgoMIV/SpeI, P210 (SERK-TMD, fragment named F67)

Investigator: Jeff, Katrin

Aim of the experiment: Preparative digestion and gelelectrophoresis of F47 (pActin, fragment named F68) with XbaI/SpeI, P202 (GFP+linker, fragment named F69) with NgoMIV/SpeI, P210 (SERK-TMD, fragment named F67) with AgeI/SpeI

Procedure:

  • Batch for preparative digestion of F47 (pActin, fragment named F68) with XbaI/SpeI
volume reagent
25 µl PCR product F47
5 µl CutSmart Buffer
1 µl XbaI(20 U/µl)
1 µl SpeI-HF (20 U/µl)
18 µl ddH2O
=50 µl TOTAL
  • Batch for preparative digestion of P202 (GFP+linker, fragment named F69) with NgoMIV/SpeI
volume reagent
20 µl plasmid P202
5 µl CutSmart Buffer
1 µl SpeI-HF(20 U/µl)
1 µl NgoMIV (20 U/µl)
23 µl ddH2O
=50 µl TOTAL
  • Batch for preparative digestion of P210 (SERK-TMD, fragment named F67) with AgeI/SpeI
volume reagent
20 µl Plasmid DNA P210
5 µl CutSmart Buffer
1 µl AgeI-HF (20 U/µl)
1 µl SpeI-HF (20 U/µl)
23 µl ddH2O
=50 µl TOTAL


  • Incubation for 3 h at 37 °C.
  • 5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batches after digestion and were loaded on a 1% agarose gel for preparative gelelectrophoresis
  • Preparative gelelectrophoresis was performed at 90 V for 60 min.

TUM13 20130608 prep Verd F47 XbaI.SpeI.png

1 kbp DNA ladder digestion of F47 (pActin, fragment named F68) with XbaI/SpeI
Fragment length as expected; band was cut out


TUM13 20130608 prep Verd P202 NgoMIV.SpeI.png


1 kbp DNA ladder digestion of P202 (GFP+linker, fragment named F69) with NgoMIV/SpeI
Fragment length as expected; band was cut out


TUM13 20130608 prep Verd P210 AgeI.SpeI.png


1 kbp DNA ladder digestion of P210 (SERK-TMD, fragment named F67) with AgeI/SpeI
Fragment length as expected; band was cut out


  • Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
  • Concentration determined with Nanodrop:
F68 pActin 10,4 ng/µl
F69 GFP+linker 13,0 ng/µl
F67 SERK-TMD 14,3 ng/µl

Preparative digestion and gelelectrophoresis of P7 (PhyB in pSB1C3, fragment named F75) with XbaI/SpeI and P8 (PhyB in pSC1C3, fragment named F76) with NgoMIV/SpeI

Investigator: Jeff, Katrin

Aim of the experiment: Preparative digestion and gelelectrophoresis of P7 (PhyB in pSB1C3, fragment named F75) with XbaI/SpeI and P8 (PhyB in pSC1C3, fragment named F76) with NgoMIV/SpeI (backbones for ligation)

Procedure:

  • Batch for preparative digestion of P7 (PhyB in pSB1C3, fragment named F75) with XbaI/SpeI
volume reagent
20 µl Plasmid P7
5 µl CutSmart Buffer
1 µl XbaI(20 U/µl)
1 µl SpeI-HF (20 U/µl)
23 µl ddH2O
=50 µl TOTAL
  • Batch for preparative digestion of P8 (PhyB in pSC1C3, fragment named F76) with NgoMIV/SpeI
volume reagent
20 µl plasmid P8
5 µl CutSmart Buffer
1 µl SpeI-HF(20 U/µl)
1 µl NgoMIV (20 U/µl)
23 µl ddH2O
=50 µl TOTAL
  • Incubation for 3 h at 37 °C.
  • before gelelectrophoresis, P7 was dephosphorylated
  • 5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batches after digestion and were loaded on a 0.5% agarose gel for preparative gelelectrophoresis
  • Preparative gelelectrophoresis was performed at 90 V for 60 min.

TUM13 20130608 prep Verd P7 XbaI.SpeI P8 NgoMIV.SpeI.png

1 kbp DNA ladder digestion of P7 (PhyB in pSB1C3, fragment named F75) with XbaI/SpeI digestion of P8 (PhyB in pSC1C3, fragment named F76) with NgoMIV/SpeI
Fragment lengths as expected; lower band was cut out Fragment lengths as expected; lower band was cut out


  • Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
  • Concentration determined with Nanodrop:
F75 dephosphorylated Backbone (P7) 39,4 ng/µl
F76 dephosphorylated Backbone (P8) 52,0 ng/µl



Dephosphorylation of F75

Investigator: Jeff, Katrin

Aim of the experiment: Dephosphorylation of cut vector pSB1C3 prevent re-ligation

Procedure:

  • the digestion product was purified with QIAquick PCR Purification Kit, Qiagen in order to change the buffer
  • Batch for the dephosphorylation of F75
volume reagent
30 µl Plasmid DNA F75 from digestion
3 µl 10X Fast AP buffer
1.3 µl FastAP Thermosensitive Alkaline

Phosphatase

=34.3 µl TOTAL
  • the reaction batch was spun briefly and incubated at 37 °C for 10 minutes
  • the reaction was stopped by heating to 75 °C for 5 minutes

Preparative digestion and gelelectrophoresis of P200 (gene synthesis 1, fragments named F70+F71) with XbaI/AgeI and P198 (gene synthesis 2, fragments named F72-F74) with XbaI/AgeI

Investigator: Jeff, Katrin

Aim of the experiment: Preparative digestion and gelelectrophoresis of P200 (gene synthesis 1, fragments named F70+F71) with XbaI/AgeI and P198 (gene synthesis 2, fragments named F72-F74) with XbaI/AgeI

Procedure:

  • Batch for preparative digestion of P200 (gene synthesis 1, fragments named F70+F71) with XbaI/AgeI
volume reagent
20 µl Plasmid P7
5 µl CutSmart Buffer
1 µl XbaI(20 U/µl)
1 µl AgeI-HF (20 U/µl)
23 µl ddH2O
=50 µl TOTAL
  • Batch for preparative digestion of P198 (gene synthesis 2, fragments named F72-F74) with XbaI/AgeII
volume reagent
20 µl Plasmid P7
5 µl CutSmart Buffer
1 µl XbaI(20 U/µl)
1 µl AgeI-HF (20 U/µl)
23 µl ddH2O
=50 µl TOTAL
  • Incubation for 3 h at 37 °C.
  • 5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batches after digestion and were loaded on a 2% agarose gel for preparative gelelectrophoresis
  • Preparative gelelectrophoresis was performed at 90 V for 60 min.

TUM13 20130608 prep Verd P200 P198 AgeI.XbaI.png

1 kbp DNA ladder digestion of P200 (gene synthesis 1, fragments named F70+F71) with XbaI/AgeI digestion of P198 (gene synthesis 2, fragments named F72-F74) with XbaI/AgeII
Fragment lengths as expected; the two lower bands were cut out (upper:F70, lower: F71) Fragment lengths as expected; all bands were cut out (upper: F72, middle: F73, lower: F74)


  • Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
  • Concentrations measured with Nano-Drop
Fragment Name Length Concentration
F70 Nanoluciferase 510 bp 9.2 ng/µl
F71 IgKappa-SigP 67 bp 3.1 ng/µl
F72 SpyCatcher 339 bp 10.8 ng/µl
F73 SERK-TMD 186 bp 8.6 ng/µl
F74 SERK-SigP 100 bp 4.8 ng/µl


Analytical digestion and gelelectrophoresis of P214-219 (Miniprep of Laccase QC IV), P179 (control before QC IV)

Investigator: Jeff, Katrin

Aim of the experiment: Analytical digestion and gelelectrophoresis of P214-219 (Miniprep of Laccase QC IV), P179 (control before QC IV)

Procedure:

  • Mastermix for analytical digestion of P214-219 (Miniprep of Laccase QC IV), P179 (control before QC IV)


  • 17.5 µl of the mastermix was added to 2.5 µl of plasmid DNA (P214-219, P179)
  • Incubation for 90 min at 37 °C.
  • digestion for 4 x 10 seconds in the microwave (600 watt), with two minute breaks in between the microwaving
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.


TUM13 20130608 anal Verd microwave P214-219,P179 NgoMVI.AgeI.png

1 kbp DNA ladder digestion of P214 (Miniprep of Laccase QC IV clone 1) with NgoMVI/AgeI digestion of P215 (Miniprep of Laccase QC IV clone 2) with NgoMVI/AgeI digestion of P216 (Miniprep of Laccase QC IV clone 3) with NgoMVI/AgeI digestion of P217 (Miniprep of Laccase QC IV clone 4) with NgoMVI/AgeI digestion of P218 (Miniprep of Laccase QC IV clone 5) with NgoMVI/AgeI digestion of P219 (Miniprep of Laccase QC IV clone 6) with NgoMVI/AgeI digestion of P179 (Miniprep of ligation product Laccase QCIII clone 2) with NgoMVI/AgeI
as expected as expected as expected as expected as expected as expected as expected

Ligation of F75+F69 (Linker+GFP into pSB1C3) and F67+F69 (Linker+GFP into TMD-pSB1C3-Suffix)

Investigator: Jeff

Aim of the experiment: Ligation of F75+F69 (Linker+GFP into pSB1C3) and F67+F69 (Linker+GFP into TMD-pSB1C3-Suffix).

Procedure:

volume reagent
2.54 µl F75 (39.4 ng/µl, ? bp)
8.30 µl F69 (13.0 ng/µl, ? bp)
6.16 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL


volume reagent
7.00 µl F67 (14.3 ng/µl, ? bp)
7.62 µl F69 (13.0 ng/µl, ? bp)
2.38 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Incubation: 1 h 22°C


Transformation of E. coli XL1 blue with F75+F69 (Linker+GFP into pSB1C3) and F67+F69 (Linker+GFP into pSB1C3-TMD-Suffix)

Investigator: Jeff

Aim of the experiment: Transformation of E. coli XL1 blue with F75+F69(Linker+GFP into pSB1C3) and F67+F69 (Linker+GFP into pSB1C3-TMD-Suffix)

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA was added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on plates containing antibiotics (CamR).

Sunday, June 9th

Miniprep of overnight cultures of transformated E. coli XL1 blue of F59+F65 (Nuclease), F59+F66 (PIF6), F59+F54 (Ig-K), F59+F55 (Luciferase), F59+F56 (SERK-SigP), F59+F57 (SERK-TMD), F59+F58 (SpyCatcher), F42+F51 (pActin), F63+F64

Investigator: Katrin

Aim of the experiment:Miniprep of overnight cultures of transformated E. coli XL1 blue of F59+F65 (Nuclease), F59+F66 (PIF6), F59+F54 (Ig-K), F59+F55 (Luciferase), F59+F56 (SERK-SigP), F59+F57 (SERK-TMD), F59+F58 (SpyCatcher), F42+F51 (pActin), F63+F64

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
  • The resulting DNA were eluted in tubes labeled as follows:
Content New Tube
F59+F65 clone 1P220
F59+F65 clone 2P221
F59+F66 clone 1P222
F59+F66 clone 2P223
F59+F56 clone 1P224
F59+F56 clone 2P225
F59+F55 clone 1P226
F59+F55 clone 2P227
F59+F57 clone 1P228
F59+F57 clone 2P229
F59+F58 clone 1P230
F59+F58 clone 2P231
F63+F64 P232
F42+F51 P233
F59+F54 clone 1P234
F59+F54 clone 2P235

Analytical digestion and gelelectrophoresis of P220-P235 ((P220-231, P233-P235 with XbaI/SpeI, P232 with MfeI/Pst1)

Investigator: Katrin

Aim of the experiment: Analytical digestion and gelelectrophoresis of P220-P235 (Minipreps)

Procedure:

  • Mastermix for analytical digestion of P220-231, P233-P235 with XbaI, SpeI
volume reagent
34 µl CutSmart Buffer
4.25 µl XbaI(20 U/µl)
4.25 µl SpeI-HF (20 U/µl)
255 µl ddH2O
=266.5 µl TOTAL
  • 17.5 µl of the mastermix was added to 2.5 µl of plasmid DNA
  • reaction batch for analytical digestion of P232 with MfeI/PstI
volume reagent
2.5 µl P232
2 µl CutSmart Buffer
0.25 µl MfeI(20 U/µl)
0.25 µl PstI-HF(20 U/µl)
15 µl ddH2O
=20 µl TOTAL


  • Incubation for 60 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min
  • for P224, P225, P228, P229, P230, P231, P234, P235,a 2% agarose gel was used, gelelectrophoresis was performed at 90 V for 45 min

20130609 anal Verd P224,225,338,229,230,231,234,235 XbaI.SpeI.png

1 kbp DNA ladder 100 bp DNA ladder digestion of P224 (SERK-SigP, clone 1) with XbaI, SpeI digestion of P225 (SERK-SigP, clone 2) with XbaI, SpeI digestion of P228 (SERK TMD, clone 1) with XbaI, SpeI digestion of P229 (SERK TMD, clone 2) with XbaI, SpeI digestion of P230 (SpyCatcher, clone 1) with XbaI, SpeI digestion of P231 (SpyCatcher, clone 2) with XbaI, SpeI digestion of P234 (Ig-K-SigP, clone 1) with XbaI, SpeI digestion of P235 (Ig-K-SigP, clone 2) with XbaI, SpeI
not as expected not as expected as expected as expected as expected as expected fragment not visible due to small size not as expected


20130609 anal Verd P220,221,222,223,226,227 XbaI.SpeI P232 MfeI.PstI P233 XbaI.SpeI.png

1 kbp DNA ladder digestion of P220 (Thermonuclease, clone 1) with XbaI, SpeI digestion of P221 (Thermonuclease, clone 2) with XbaI, SpeI digestion of P222 (PIF6, clone 1) with XbaI, SpeI digestion of P223 (PIF6, clone 2) with XbaI, SpeI digestion of P226 (Luciferase, clone 1) with XbaI, SpeI digestion of P227 (Luciferase, clone 2) with XbaI, SpeI digestion of P232 (miniMCS) with MfeI/PstI digestion of P233 (pActin) with XbaI, SpeI
not as expected not as expected as expected as expected as expected as expected not as expected as expected

Picking of F67+F69 (SERK-TMD+linker GFP)

Investigator: Katrin

Aim of the study: Picking of F67+F69 (SERK-TMD+linker GFP)

Operational sequence:

  • Picking and overnight culture after standard laboratory's protocol. (5 µl CamR and 5 ml LB-medium)
  • 3 colonies were picked for every Transformation product.
  • There were no colonies on the plates for ligations F68+F75 (pActin, pSB1C3),

(probably due to dephosphorylation)

Preparative digestion and gelelectrophoresis of P220 (thermonuclease, fragment corrupted) with NgoMIV/SpeI, P160 (NLS, fragment named F77) with AgeI/SpeI, P40 (GSAT linker, fragment named F79) with NgoMIV/SpeI, P7 (PhyB, fragment named F78) with AgeI/SpeI

Investigator: Katrin

Aim of the experiment: Preparative digestion and gelelectrophoresis of P220 (thermonuclease, fragment corrupted) with NgoMIV/SpeI, P160 (NLS, fragment named F77) with AgeI/SpeI, P40 (GSAT linker, fragment named F79) with NgoMIV/SpeI, P7 (PhyB, fragment named F78) with AgeI/SpeI

Procedure:

  • Batch for preparative digestion of P220 (thermonuclease, fragment corrupted) with NgoMIV/SpeI
volume reagent
20 µl P220
5 µl CutSmart Buffer
1 µl NgoMIV(20 U/µl)
1 µl SpeI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P160 (NLS, fragment named F77) with AgeI/SpeI
volume reagent
20 µl plasmid P160
5 µl CutSmart Buffer
1 µl SpeI-HF(20 U/µl)
1 µl AgeI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P40 (GSAT linker, fragment named F79) with NgoMIV/SpeI
volume reagent
20 µl plasmid P40
5 µl CutSmart Buffer
1 µl SpeI-HF(20 U/µl)
1 µl NgoMIV (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P7 (PhyB, fragment named F78) with AgeI/SpeI
volume reagent
20 µl plasmid P40
5 µl CutSmart Buffer
1 µl SpeI-HF(20 U/µl)
1 µl AgeI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Incubation for 2 h at 37 °C.
  • 4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches containing P220 and P40 after digestion and were loaded on a 1% agarose gel for preparative gelelectrophoresis
  • the vector backbones where only a few base pairs were cut out were purified via PCR-purification
  • Preparative gelelectrophoresis was performed at 90 V for 60 min.

20130609 prep Verd P220, P40 NgoMIV.SpeI.png

digestion of P220 (thermonuclease, fragment corrupted) with NgoMIV/SpeI 1 kbp DNA ladder digestion of P40 (GSAT linker, fragment named F79) with NgoMIV/SpeI
not as expected, band was discarded as expected, lower band (108 bp) was cut out
  • the band was extracted with QIAquick Gel Extraction Kit, QIAGEN.

PCR Purification of vector backbones P7 (PhyB in pSB1C3, fragment F78), P160 (NLS in pSB1C3, fragment F77)

Investigator: Katrin

Aim of the study: PCR Purification of vector backbones P7 (PhyB in pSB1C3, fragment F78), P160 (NLS in pSB1C3, fragment F77)

  • in order to get rid of the few base pairs that were cut out during digestion, the digestion batches were purified 
with QIAquick PCR Purification Kit, Qiagen
  • the yiedls were 56.2 ng/µl for F77 and 277,6 ng/µl for F78

Ligation of F78+F79(PhyB+linker) and F59+F71 (pSB1C3+Ig-K)

Investigator: Katrin Aim of the experiment: Ligation of F78+F79(PhyB+linker) and F59+F71 (pSB1C3+Ig-K)

Procedure:

  • Ligation batch for F78+F79(PhyB+linker)
volume reagent
0.36 µl F78 (277.6 ng/µl, 4807 bp)
1.77 µl P79 (3.8 ng/µl, 108 bp)
14.87 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F59+F71 (pSB1C3+Ig-K)
volume reagent
1.5 µl F59 (66.7 ng/µl, 2086 bp)
3.12 µl P71 (3.1 ng/µl, 67 bp)
12.38 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
  • The ligation was performed for 1 hour at room temperature.

Transformation of E. coli XL1 blue with F78+F79(PhyB+linker) and F59+F71 (pSB1C3+Ig-K)

Investigator: Katrin

Aim of the experiment: Transformation of E. coli XL1 blue with F78+F79(PhyB+linker) and F59+F71 (pSB1C3+Ig-K) and negative controls F78 and F59

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA was added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on plates containing antibiotics (CamR).

PCR of P192 (Thermonuclease, O64 & O65)

Investigator: Katrin

Aim of the experiment: PCR of P192 (Thermonuclease, O64 & O65)

Procedure:

Operational sequence:

  • PCR reaction mixture
volume reagent
10 µl 5x OneTaq Standard Reaction Buffer
1 µl 10 mM dNTPs
1 µl 10 µM Forward Primer O64 (NucA_fw)
1 µl 10 µM Reverse Primer O65 (NucA_rv)
0.25 µL OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl 1:1000 dilution of plasmid DNA (P192)
35.75 µL ddH2O Water
=50 µL TOTAL
  • Mix with pipette
  • The gradient PCR program was performed after following scheme with following conditions:
Initial denaturation 94 °C 30 s
30 cycles 94 °C 30 s
55 °C 60 s
68 °C 30 s
Final extension 68 °C 5 min
Hold 4 °C infinite

Week 8

Monday, June 10rd

Analytical Gel of PCR of Thermonuclease (P192)

Investigator: Florian

Aim of the study: Analysis of PCR of Thermonuclease (P192); supposed letgth: 447 bp

TUM13 20130610 anal gel PCR P192.png

Lane: DNA ladder 1kb P192 PCR product DNA ladder 1kb
Result: - as expected -

PCR Purification of Thermonuclease (P192)

Investigator: Andi

Aim of the study: PCR Purification of Thermonuclease (P192)

  • After the PCR the batch was purified with QIAquick PCR Purification Kit, Qiagen
  • Eluation in 35 µl EB buffer -> F80

Miniprep of F67+F69 Ligation (Linker+GFP in Suffix of TMD)

Investigator: Johanna

Aim of the study: Preparation of DNA of the F67+F69 Ligation from 09.06.13 (Linker+GFP in Suffix of TMD)

Procedure: according to MiniPrep Kit QIAGEN; measurement of concentration (NanoDrop)

  • Eluation in 35 µl EB buffer each
Plamsid Concentration
P236 108.0 ng/µl
P237 116.0 ng/µl
P238 165.4 ng/µl

Picking of F78 + F79 (PhyB + Linker) and F59 + F71 (pSB1C3 + lgkappa)

Investigator: Johanna

Aim of the study: Picking of F78 + F79 (PhyB + Linker) and F59 + F71 (pSB1C3 + lgkappa)

Operational sequence:

  • Picking and overnight culture after standard laboratory's protocol. (4 µl CamR and 4 ml LB-medium)
  • 3 colonies were picked for every Transformation product.

Sequencing of P150 (FluA), P233 (pActin), P213 (Spycatcher) and F67+F69 Ligation (Linker+GFP + TMD)

Investigator: Johanna

Aim of the study: Sequencing of P150 (FluA), P233 (pActin), P213 (Spycatcher) and F67+F69 Ligation (Linker+GFP + TMD)

Procedure:Eurofins SmartSeq Kit protocol (15 µl plasmid DNA (50 - 100 ng), 2 µl sequencing primer).

# P150 P233 P213 P236 P237
Name FluA pActin Spycatcher Linker+GFP+TMD clone1 Linker+GFP+TMD clone2
O3 (forward) FR01002308 FR01002307 FR01002305 FR01002299 FR01002298
O4 (reverse) n.a. FR01002306 n.a. FR01002297 FR01002311

PCR of BPul Laccase P219 + Analytical Gelelectrophoresis

Investigator: Andi, Johanna

Aim of the experiment: PCR of Bpul Laccase P219.

Procedure:

Operational sequence:

  • PCR reaction mixture
volume reagent
10 µl 5x OneTaq Reaction Standard Reaction Buffer
1 µl 10 mM dNTPs
1 µl 10 µM Forward Primer (P219:O24 (Bpul_for))
1 µl 10 µM Reverse Primer (P143:O25 (Bpul_rev))
0.25 µL OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl Plasmid DNA (P219)- 1:1000 dilution
35.75 µL ddH2O Water
=50 µL TOTAL
  • Mix with pipette
  • The PCR program was performed after following scheme:
Initial denaturation 94 °C 30 s
30 cycles 94 °C 30 s
55 °C 60 s
68 °C 90 s
Final extension 68 °C 5 min
Hold 4 °C infinite
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
  • The resulting fragment was called F82
  • Further on an analytif Gelelectrophoresis was performed to be sure that the expected fragment was amplified.

TUM13 20130610 PCR P219.png

DNA ladder 1kb P219 PCR product
  • As we can see, the fragment has the expected length of about 1600 BP.

Preparative digestion of F80 (PCR purification of Thermonuclease) with XbaI/AgeI

Investigator: Florian, Johanna

Aim of the experiment: Preparative digestion of F80 (PCR purification of Thermonuclease) with XbaI/AgeI.

Procedure:

  • Batch for preparative digestion of F80 (PCR purification of Thermonuclease) with XbaI/AgeI.
volume reagent
25 µl F80
5 µl CutSmart Buffer
1 µl AgeI (20 U/µl)
1 µl XbaI (20 U/µl)
18 µl ddH2O
=50 µl TOTAL
  • Incubation for 2.5 h at 37 °C.
  • The digested fragment was purified via PCR-purification (Qiagen-kit)
  • A yield of 26.2 ng/µl could be measured for F81

Ligation of F59 + F81 (Thermonuclease in pSB1C3)

Investigator: Florian, Johanna

Aim of the experiment: Ligation of F59 + F81 (Thermonuclease in pSB1C3).

Procedure:

  • Ligation batch for F59 + F81 (Thermonuclease in pSB1C3)
volume reagent
1.5 µl F59 (66.7 ng/µl, 2086 bp)
2.45 µl P81 (26.2 ng/µl, 447 bp)
13.55 µl ddH2O
2 µl T4 ligase buffer (10x)
0.5 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
  • The ligation was performed for 1 hour at room temperature.

Transformation of E. coli XL1 blue with F59+F81(Thermonuclease in pSB1C3)

Investigator: Katrin

Aim of the experiment: Transformation of E. coli XL1 blue with F59+F81(Thermonuclease in pSB1C3) and the negative control F59.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA was added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Addition of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspensions were plated on plates containing antibiotics (CamR).
  • The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on plates containing antibiotics (CamR).

Preparative digestion and purification of F82 (PCR product of Bpul Laccase) with XbaI/AgeI-HF and PCR purification kit of Qiagen

Investigator: Andi, Johanna

Aim of the experiment: Preparative digestion and purification of F82 (Bpul Laccase) with XbaI+AgeI and PCR purification kit of Qiagen

Procedure:

  • Batch for preparative digestion of F82 with XbaI/AgeI
volume reagent
25 µl F82
5 µl CutSmart Buffer
1 µl AgeI-HF(20 U/µl)
1 µl XbaI (20 U/µl)
18 µl ddH2O
=50 µl TOTAL
  • Incubation for 2.5 h at 37 °C.
  • the digested fragment was purified via PCR-purification
  • the resulting fragment was called F83 (F82 digested with XbaI+AgeI)

Ligation of F83(PCR product of Bpul Laccase digested with AgeI-HF + XbaI)and F59 (psB1C3 digested with AgeI-HF + XbaI)

Investigator: Andi, Johanna

Aim of the experiment: Ligation of F83(PCR product of Bpul Laccase digested with AgeI-HF + XbaI)and F59 (psB1C3 digested with AgeI-HF + XbaI)

Procedure:

  • Ligation batch for F83+F59 (psB1C3+Bpul Laccase)
volume reagent
15.5 µl F83 (9.2 ng/µl, 1600 bp)
1.5 µl F59 (66.7 ng/µl, 2100 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
  • The ligation was performed over night at 16°C.

Tuesday, June 11th

Transformation of E. coli XL1 blue with ligation products F59+F81 and F59+F83

Investigator: Louise, Andi

Aim of the experiment: Transformation of E. coli XL1 blue with ligation products F59+F81 and F59+F83.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA was added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on new chlorampenicol plates.


Miniprep of overnight cultures of transformated E. coli XL1 blue with F78+F79 (PhytochromB +Linker) and F59+F71 (IgKappa)

Investigator: Louise, Johanna

Aim of the experiment:Miniprep of overnight cultures of transformated E. coli XL1 blue with F78+F79 (PhytochromB +Linker) and F59+F71 (IgKappa).

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
  • The resulting DNA were eluted in tubes labeled as follows:
Content New Tube
F78+F79 (PhytochromB +Linker) clone 1P239
F78+F79 (PhytochromB +Linker) clone 2P240
F78+F79 (PhytochromB +Linker) clone 3P241
F59+F71 (IgKappa) clone 4P242
F59+F71 (IgKappa) clone 5P243
F59+F71 (IgKappa) clone 6P244

Analytical digestion and gelelectrophoresis of P239-P241 (PhytochromB+Linker) and P242-P244 (IgKappa) with XbaI and AgeI

Investigator: Louise, Rosario, Florian

Aim of the experiment: Analytical digestion and gelelectrophoresis of P239-P241 (PhytochromB+Linker) and P242-P244 (IgKappa) with XbaI and AgeI

Procedure:

  • Batch
volume reagent
2 µl CutSmart Buffer (10x)
0.25 µl AgeI
0.25 µl XbaI
15 µl ddH2O
2.5 µl Plasmid DNA (P293-P244)
=20 µl TOTAL
  • Incubation for 60 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min
  • For P242-P244 a 2% agarose gel was used and for P293-P241 a 0.5% agarose gel was used.

TUM13 20130611 anal gel P242-P244 XbaI AgeI.png

Lane: Digestion of P242 (IgK) with XbaI & AgeI, clone 1 Digestion of P243 (IgK) with XbaI & AgeI, clone 2 Digestion of P244 (IgK) with XbaI & AgeI, clone 3 DNA ladder 100bp
Result: as expected as expected as expected -


TUM13 20130611 anal gel P239-P241 XbaI AgeI.png

Lane: Digestion of P239 (PhyB+Linker) with XbaI & AgeI, clone 1 Digestion of P240 (PhyB+Linker) with XbaI & AgeI, clone 2 Digestion of P241 (PhyB+Linker) with XbaI & AgeI, clone 3 DNA ladder 1kb
Result: failed as expected as expected -


Preparative digestion and gelelectrophoresis of P204 (IgKappa-SigP, fragment named F90) with AgeI/SpeI, P157 (EreB, fragment named F86) with NgoMIV/SpeI, P208 (NanoLuc, fragment named F87) with NgoMIV/SpeI, P213 (Spycatcher, fragment named F88) with AgeI/SpeI and P170 (Spytag, fragment named F89) with NgoMIV/SpeI

Investigator: Johanna, Rosario, Flo

Aim of the experiment: Preparative digestion and gelelectrophoresis of P204 (IgKappa-SigP, fragment named F90) with AgeI/SpeI, P157 (EreB, fragment named F86) with NgoMIV/SpeI, P208 (NanoLuc, fragment named F87) with NgoMIV/SpeI, P213 (Spycatcher, fragment named F88) with AgeI/SpeI and P170 (Spytag, fragment named F89) with NgoMIV/SpeI

Procedure:

  • Batch for preparative digestion of P204 (IgKappa-SigP, fragment named F90) with AgeI/SpeI
volume reagent
20 µl P204
5 µl CutSmart Buffer
1 µl AgeI(20 U/µl)
1 µl SpeI-HF (20 U/µl)
23 µl ddH2O
=50 µl TOTAL
  • Batch for preparative digestion of P157 (EreB, fragment named F86) with NgoMIV/SpeI
volume reagent
20 µl P157
5 µl CutSmart Buffer
1 µl NgoMIV(20 U/µl)
1 µl SpeI-HF (20 U/µl)
23 µl ddH2O
=50 µl TOTAL
  • Batch for preparative digestion of P208 (NanoLuc, fragment named F87) with NgoMIV/SpeI
volume reagent
20 µl P208
5 µl CutSmart Buffer
1 µl NgoMIV(20 U/µl)
1 µl SpeI-HF (20 U/µl)
23 µl ddH2O
=50 µl TOTAL
  • Batch for preparative digestion of P213 (Spycatcher, fragment named F88) with NgoMIV/SpeI
volume reagent
20 µl P213
5 µl CutSmart Buffer
1 µl NgoMIV(20 U/µl)
1 µl SpeI-HF (20 U/µl)
23 µl ddH2O
=50 µl TOTAL
  • Batch for preparative digestion of P170 (Spytag, frament named F89) with NgoMIV/SpeI
volume reagent
20 µl P170
5 µl CutSmart Buffer
1 µl NgoMIV(20 U/µl)
1 µl SpeI-HF (20 U/µl)
23 µl ddH2O
=50 µl TOTAL
  • Incubation for 2.5 h at 37 °C.
  • 5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batches after digestion and were loaded on a 1% agarose gel (P204, P157, P208, P213) and 2 % agarose gel (P170) for preparative gelelectrophoresis
  • Preparative gelelectrophoresis was performed at 90 V for 60 min.

20130611 prep Verd P204, P157 NgoMIV.SpeI.png

1 kbp DNA ladder digestion of P204 (IgKappa-SigP) with AgeI/SpeI 100 bp DNA ladder (Generuler) digestion of P157 (EreB) with NgoMIV/SpeI
as expected, lower band was cut out as expected, lower band was cut out

20130611 prep Verd P208, P1213 NgoMIV.SpeI.png

1 kbp DNA ladder digestion of P208 (Nanoluc) with NgoMIV/SpeI 100 bp DNA ladder (Generuler) digestion of P213 (Spycatcher) with NgoMIV/SpeI
as expected, lower band was cut out as expected, lower band was cut out

20130611 prep Verd P170 AgeI.SpeI.png

100 bp DNA ladder (Generuler) digestion of P170 (Spytag) with NgoMIV/SpeI
as expected, lower band was cut out
  • the band was extracted with QIAquick Gel Extraction Kit, QIAGEN.

Ligation of F90+F86 (IgKappa-SigP + EreB), F90+F87 (IgKappa-SigP + Nanoluciferase), F90+F88 (IgKappa-SigP + SpyCatcher), F90+F89 (IgKappa-SigP + SpyTag) and negativ control of F90 (IgKappa-SigP)

Investigator: Johanna

Aim of the experiment: Ligation of F90+F86 (IgKappa-SigP + EreB), F90+F87 (IgKappa-SigP + Nanoluciferase), F90+F88 (IgKappa-SigP + SpyCatcher), F90+F89 (IgKappa-SigP + SpyTag) and negativ control of F90 (IgKappa-SigP)

Procedure:

  • Ligation batch for F90+F86 (IgKappa-SigP + EreB)
volume reagent
5.7 µl F86 (31.0 ng/µl, 1254 bp)
6.5 µl F90 (15.3 ng/µl, 2144 bp)
4.8 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F90+F87 (IgKappa-SigP + Nanoluciferase)
volume reagent
7.0 µl F87 (10.2 ng/µl, 510 bp)
6.5 µl F90 (15.3 ng/µl, 2144 bp)
3.5 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F90+F88 (IgKappa-SigP + SpyCatcher)
volume reagent
6.3 µl F88 (8.7 ng/µl, 339 bp)
6.5 µl F90 (15.3 ng/µl, 2144 bp)
4.2 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F90+F89 (IgKappa-SigP + SpyTag)
volume reagent
10.5 µl F89 (8.7 ng/µl, 39 bp)
6.5 µl F90 (15.3 ng/µl, 2144 bp)
0.0 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Negative control of F 90 (IgKappa-SigP) was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
  • The ligation was performed for 1 hour at room temperature.

PCR of pActin (P143) and Thermonuclease (P192) to find the best conditions for PCR + Measurement via Nanodrop + Analytical Gelelectrophoresis to get to know about it!

Investigator: Andi

Aim of the experiment: PCR of pActin (P143) and Thermonuclease (P192) to find the best conditions for PCR + Measurement via Nanodrop + Analytical Gelelectrophoresis to get to know about it!

Procedure:

Operational sequence:

  • The PCR for P143 (pActin) was performed three times under different conditions
  • First batch - PCR reaction mixture
volume reagent
10 µl 5x OneTaq Reaction Standard Reaction Buffer
1 µl 10 mM dNTPs
1 µl 10 µM Forward Primer (P143:O62 (pAct_for))
1 µl 10 µM Reverse Primer (P143:O63 (pAct_rev))
0.25 µL OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl Plasmid DNA (P143)- 1:1000 dilution
35.75 µL ddH2O Water
=50 µL TOTAL
  • Second batch - PCR reaction mixture
volume reagent
10 µl 5x OneTaq GC Reaction Buffer
1 µl 10 mM dNTPs
1 µl 10 µM Forward Primer (P143:O62 (pAct_for))
1 µl 10 µM Reverse Primer (P143:O63 (pAct_rev))
0.25 µL OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl Plasmid DNA (P143)- 1:1000 dilution
35.75 µL ddH2O Water
=50 µL TOTAL
  • Third batch - PCR reaction mixture
volume reagent
10 µl 5x Herculase Fusion Polymerase reaction buffer
2.5 µl 10 mM dNTPs
1.25 µl 10 µM Forward Primer (P143:O62 (Bpul_for))
1.25 µl 10 µM Reverse Primer (P143:O63 (Bpul_rev))
1 µL Herculase II Fusion Polymerase
2 µl Plasmid DNA (P143)- 1:1000 dilution
0.5 µl DMSO
35.75 µL ddH2O Water
=50 µL TOTAL
  • The content of all batches has been mixed with a pipette
  • The PCR program was performed after following scheme:
Initial denaturation 94 °C 30 s
30 cycles 94 °C 30 s
51 °C 60 s
68 °C 90 s
Final extension 68 °C 5 min
Hold 4 °C infinite


  • The PCR for P192(Thermonuclease) was performed three times under different conditions
  • First batch - PCR reaction mixture
volume reagent
10 µl 5x OneTaq Reaction Standard Reaction Buffer
1 µl 10 mM dNTPs
1 µl 10 µM Forward Primer (P192:O64 (NucA_fw))
1 µl 10 µM Reverse Primer (P192:O65(NucA_rev))
0.25 µL OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl Plasmid DNA (P192)- 1:1000 dilution
35.75 µL ddH2O Water
=50 µL TOTAL
  • Second batch - PCR reaction mixture
volume reagent
10 µl 5x OneTaq GC Reaction Buffer
1 µl 10 mM dNTPs
1 µl 10 µM Forward Primer (P192:O64 (NucA_fw))
1 µl 10 µM Reverse Primer (P192:O65(NucA_rev))
0.25 µL OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl Plasmid DNA (P192)- 1:1000 dilution
35.75 µL ddH2O Water
=50 µL TOTAL
  • Third batch - PCR reaction mixture
volume reagent
10 µl 5x Herculase II DNA Fusion Polymerase reaction Buffer
2.5 µl 10 mM dNTPs
1.25 µl 10 µM Forward Primer (P192:O64 (NucA_fw))
1.25 µl 10 µM Reverse Primer (P192:O65(NucA_rev))
1 µL Herculase II Fusion DNA Polymerase
3 µl Plasmid DNA (P192)- 1:1000 dilution
0.5 µL DMSO
30.5 µL ddH2O Water
=50 µL TOTAL
  • The PCR program of the Thermonuclease (P192) was performed after following scheme:
Initial denaturation 94 °C 30 s
30 cycles 94 °C 30 s
54 °C 60 s
68 °C 40 s
Final extension 68 °C 5 min
Hold 4 °C infinite
  • Mix the content of the batches with pipette
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
  • The concentrations of all PCR products have been measured
P143 - Batch #1 - PCR product - 5.9 ng/µl P143 - Batch #2 - PCR product - 40.5 ng/µl P143 - Batch #3 - PCR product - 12.4 ng/µl P192 - Batch #1 - PCR product - 39.7 ng/µl P192 - Batch #2 - PCR product - 23.1 ng/µl P192 - Batch #3 - PCR product - 82.5 ng/µl
  • As we can see, the highest concentration of PCR product seems to be in P143 - Batch #2 and P192 - Batch #3.
  • Further on an analytic Gelelectrophoresis was performed to be sure that the expected fragment was amplified.

TUM13 20130611 PCR P143 TaqStand TaqGC Herc P192 TaqStand TaqGC Herc.png

DNA ladder 1kb P143 - Batch #1 - PCR product P143 - Batch #2 - PCR product P143 - Batch #3 - PCR product P192 - Batch #1 - PCR product P192 - Batch #2 - PCR product P192 - Batch #3 - PCR product
  • As we can see, the fragment has the expected length of about 1600 BP referring to the PCR product of P143 and about XXX BP referring to the product of P192.
  • Further on the analytical gel confirms the output of the nano drop.


Preparative digestion of PCR reaction batch #2 of P143 (pActin) XbaI/PstI and PCR reaction batch #3 of P192 with XbaI/AgeI

Investigator: Andi

Aim of the experiment: Preparative digestion of PCR reaction batch #2 of P143 (pActin) XbaI/PstI and PCR reaction batch #3 of P192 with XbaI/AgeI

Procedure:

  • Batch for preparative digestion of PCR reaction batch #2 of P143 (pActin) with XbaI/PstI.
volume reagent
24 µl PCR reaction batch #2 of P143
5 µl CutSmart Buffer
1 µl PstI #1 (20 U/µl)
1 µl PstI #2 (20 U/µl)
1 µl XbaI (20 U/µl)
18 µl ddH2O
=50 µl TOTAL
  • I used 1 µl of both PstI batches because one seems to be broken
  • Incubation for 2.5 h at 37 °C.
  • The digested fragment was purified via PCR-purification (Qiagen-kit)
  • A yield of 16.9 ng/µl could be measured for the resulting F84


  • Batch for preparative digestion of PCR reaction batch #3 of P192 (Thermonuclease) with XbaI/AgeI-HF.
volume reagent
24 µl PCR reaction batch #2 of P143
5 µl CutSmart Buffer
1 µl AgeI-HF (20 U/µl)
1 µl XbaI (20 U/µl)
19 µl ddH2O
=50 µl TOTAL
  • Incubation for 2.5 h at 37 °C.
  • The digested fragment was purified via PCR-purification (Qiagen-kit)
  • A yield of 46.2 ng/µl could be measured for the resulting F85

Ligation of F84(Digestion of PCR product (GC rich buffer) of P143 (pActin) with PstI/XbaI) and F42 (psB1C3 digested with PstI/XbaI) and Ligation of F85(Digestion of PCR product (Herculase II) of P192 (Thermonuclease) with XbaI/AgeI and F59 (psB1C3 digested with AgeI-HF + XbaI)

Investigator: Andi

Aim of the experiment: Ligation of F84(Digestion of PCR product (GC rich buffer) of P143 (pActin) with PstI/XbaI) with F42 (psB1C3 digested with PstI/XbaI) and Ligation of F85(Digestion of PCR product (Herculase II) of P192 (Thermonuclease) with XbaI/AgeI-HF) with F59 (psB1C3 digested with AgeI-HF/XbaI)

Procedure:

  • Ligation batch for F84+F42 (psB1C3+pActin)
volume reagent
12.6 µl F84 (16.9 ng/µl, 1500 bp)
1.4 µl F42 (73.5 ng/µl, 2100 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
4 µl ddH2O
=20 µl TOTAL
  • Ligation batch for F85+F59 (psB1C3+Thermonuclease)
volume reagent
1.6 µl F85 (46.2 ng/µl, 500 bp)
1.5 µl F59 (66.7 ng/µl, 2100 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
13.9 µl ddH2O
=20 µl TOTAL
  • Negative control was also prepared for both batches. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
  • The ligation was performed over night at 16°C.

Transformation of E. coli XL1 blue with plasmids P157, P208 and P213

Investigator: Florian

Aim of the experiment: Transformation of E. coli XL1 blue with plasmids P157, P208 and P213.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA was added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on new chlorampenicol plates.

Wednesday, June 12th

Transformation of E. coli XL1 blue with F42+F84, F90+F86, F90+F87, F90+F88, F90+F89, F59+F85

Investigator: Rosario

Aim of the experiment: Transformation of E. coli XL1 blue with F42+F84, F90+F86, F90+F87, F90+F88, F90+F89, F59+F85

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA was added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • Centrifugation for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated on new chlorampenicol plates.

Preparative digestion and gelelectrophoresis of P240 (PhyB + Linker) with AgeI/SpeI, P244 (IgKappa) with AgeI/SpeI, P197 (XylE) with NgoMIV/SpeI, P150 (FluA) with NgoMIV/SpeI, P126 (N-TEV) with AgeI/SpeI, P127 (C-TEV) with NgoMIV/SpeI, P168 (PIF3) with NgoMIV/SpeI and P222 (PIF6) with NgoMIV/SpeI

Investigator: Louise, Florian

Aim of the experiment: Preparative digestion and gelelectrophoresis of P240 (PhyB + Linker) with AgeI/SpeI, P244 (IgKappa) with AgeI/SpeI, P197 (XylE) with NgoMIV/SpeI, P150 (FluA) with NgoMIV/SpeI, P126 (N-TEV) with AgeI/SpeI, P127 (C-TEV) with NgoMIV/SpeI, P168 (PIF3) with NgoMIV/SpeI and P222 (PIF6) with NgoMIV/SpeI

Procedure:

  • Batch for preparative digestion of P240 (PhyB + Linker) with AgeI/SpeI
volume reagent
20 µl P240
4 µl CutSmart Buffer
1 µl AgeI(20 U/µl)
1 µl SpeI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P244 (IgKappa) with AgeI/SpeI
volume reagent
20 µl P244
4 µl CutSmart Buffer
1 µl AgeI(20 U/µl)
1 µl SpeI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P197 (XylE) with NgoMIV/SpeI
volume reagent
20 µl P197
4 µl CutSmart Buffer
1 µl NgoMIV(20 U/µl)
1 µl SpeI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P150 (FluA) with NgoMIV/SpeI
volume reagent
20 µl P150
4 µl CutSmart Buffer
1 µl NgoMIV(20 U/µl)
1 µl SpeI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P126 (N-TEV) with AgeI/SpeI
volume reagent
20 µl P126
4 µl CutSmart Buffer
1 µl AgeI(20 U/µl)
1 µl SpeI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P127 (C-TEV) with NgoMIV/SpeI
volume reagent
20 µl P127
4 µl CutSmart Buffer
1 µl NgoMIV(20 U/µl)
1 µl SpeI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P168 (PIF3) with NgoMIV/SpeI
volume reagent
20 µl P168
4 µl CutSmart Buffer
1 µl NgoMIV(20 U/µl)
1 µl SpeI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P222 (PIF6) with NgoMIV/SpeI
volume reagent
20 µl P222
4 µl CutSmart Buffer
1 µl NgoMIV(20 U/µl)
1 µl SpeI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Incubation for 2.5 h at 37 °C.
  • 4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and were loaded on a 1% agarose gel for preparative gelelectrophoresis
  • Preparative gelelectrophoresis was performed at 90 V for 60 min.

TUM13 20130612 PrepGel P126 AgeI.SpeI P127 NgoMIV.SpeI P150 NgoMIV.SpeI.png

1 kbp DNA ladder digestion of P126 (N-TEV) with AgeI/SpeI digestion of P127 (C-TEV) with NgoMIV/SpeI digestion of P150 (FluA) with NgoMIV/SpeI
as expected, upper band was cut out as expected, lower band was cut out as expected, lower band was cut out

TUM13 20130612 PrepGel P168 NgoMIV.SpeI P197 NgoMIV.SpeI P222 NgoMIV.SpeI.png

1 kbp DNA ladder digestion of P168 (PIF3) with NgoMIV/SpeI digestion of P197 (XylE) with NgoMIV/SpeI digestion of P222 (PIF6) with NgoMIV/SpeI
as expected, lower band was cut out as expected, lower band was cut out as expected, lower band was cut out

TUM13 20130612 PrepGel P244 AgeI.SpeI P240 AgeI.SpeI.png

100 bp DNA ladder (Generuler) digestion of P244 (IgKappa) with AgeI/SpeI digestion of P240 (PhyB + Linker) with AgeI/SpeI 1 kbp DNA ladder
as expected, upper band was cut out as expected, upper band was cut out
  • the DNA was extracted from the gel with QIAquick Gel Extraction Kit, QIAGEN.
  • the fragments received the following names:
P126 (N-TEV) F93
P127 (C-TEV) F94
P150 (FluA) F95
P168 (PIF3) F96
P197 (XylE) F97
P222 (PIF6) F98
P240 (PhyB + Linker) F91
P244 (IgKappa) F92

Transformation of E. coli XL1 blue with plasmids P126 (N-TEV), P127 (C-TEV), P222 (PIF6), P240 (PhyB+Linker) and P244 (IgKappa)

Investigator: Florian

Aim of the experiment: Transformation of E. coli XL1 blue with plasmids P126 (N-TEV), P127 (C-TEV), P222 (PIF6), P240 (PhyB+Linker) and P244 (IgKappa).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA was added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension were plated on chloramphenicol plates.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on new chlorampenicol plates.

Picking of P157, P208, P213, F59 + F81 (Thermonuclease in pSB1C3) and F59 + F83 (Laccase in pSB1C3)

Investigator: Jeff

Aim of the study: Picking of P157, P208, P213, F59 + F81 (Thermonuclease in pSB1C3) and F59 + F83 (Laccase in pSB1C3).

Operational sequence:

  • Picking and overnight culture after standard laboratory's protocol. (4 µl CamR and 4 ml LB-medium)
  • 2 colonies were picked for every ligation product and 1 colony was picked for every transformation product.


Ligation of E. coli XL1 blue with F92+F89, F92+F87, F92+F86, F92+F88, F92+F97, F90+F97, F91+F94, F93+F79 and their negative controls

Investigator: Jeff, Flo, Louise, Christopher

Aim of the experiment: Ligation of E. coli XL1 blue with F92+F89, F92+F87, F92+F86, F92+F88, F92+F97, F90+F97, F91+F94, F93+F79 and their negative controls

Procedure:

  • Ligation batch for F92+F89 :
volume reagent
0.86 µl F92 (116.7 ng/µl, 2153 bp)
16.14 µl F89 (6.8 ng/µl, 39 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
0.0 µl ddH2O
=20 µl TOTAL
  • Ligation batch for F92+F87 :
volume reagent
0.86 µl F92 (116.7 ng/µl, 2153 bp)
7.0 µl F87 (10.2 ng/µl, 510 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
9.14 µl ddH2O
=20 µl TOTAL
  • Ligation batch for F92+F86:
volume reagent
0.86 µl F92 (116.7 ng/µl, 2153 bp)
5.7 µl F86 (31.0 ng/µl, 1260 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
10.44 µl ddH2O
=20 µl TOTAL
  • Ligation batch for F92+F88 :
volume reagent
0.86 µl F92 (116.7 ng/µl, 2153 bp)
5.4 µl F88 (8.7 ng/µl, 339 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
10.74 µl ddH2O
=20 µl TOTAL
  • Ligation batch for F92+F97:
volume reagent
0.86 µl F92 (116.7 ng/µl, 2153 bp)
6.6 µl F97 (19.4 ng/µl, 918 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
9.5 µl ddH2O
=20 µl TOTAL
  • Ligation batch for F90+F97:
volume reagent
6.5 µl F90 (15.3 ng/µl, 2144 bp)
6.6 µl F97 (19.4 ng/µl, 918 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
3.9 µl ddH2O
=20 µl TOTAL
  • Ligation batch for F91+F94:
volume reagent
0.63 µl F91 (159 ng/µl, 4129 bp)
4.6 µl F94 (5.6 ng/µl, 354 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
11.77 µl ddH2O
=20 µl TOTAL
  • Ligation batch for F93+F79:
volume reagent
3.6 µl F93 (27.4 ng/µl, 2440 bp)
3.5 µl F79 (3.8 ng/µl, 108 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
9.9 µl ddH2O
=20 µl TOTAL
  • Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
  • The ligation was performed at room temperature for 1 hour

Transformation of E. coli XL1 blue with F92+F89, F92+F87, F92+F86, F92+F88, F92+F97, F90+F97, F91+F94, F93+F79 and their negative controls

Investigator: Jeff, Florian

Aim of the experiment: Transformation of E. coli XL1 blue with F92+F89, F92+F87, F92+F86, F92+F88, F92+F97, F90+F97, F91+F94, F93+F79 and their negative controls.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA was added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • Centrifugation for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated on new chloramphenicol plates.

Thursday, June 13th

QuikChange mutagenesis to remove forbidden restriction sites - SDMIV - of P250, P251, P219 (Laccase), P163 (NPT-Cassette)

Investigator: Andi

Aim of the experiment: Removal of forbidden restriction site from P251 (Laccase) and P163 (NPT-Cassette).

Procedure: Quickchange - PCR

Reaction batch for P219 (Laccase)

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid template (P251 - 1:20 dilution) need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl 1:10 dilution of O74 (10 µM)
1 µl 1:10 dilution of O75 (10 µM)
18 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)

Reaction batch for P163 (NPT-Cassette)

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid template (P163 - 1:60 dilution) need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl 1:10 dilution of O70 (10 µM)
1 µl 1:10 dilution of O71 (10 µM)
18 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)


Reaction batch for P250 (Laccase)

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid template (P250 - 1:20 dilution) need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl 1:10 dilution of O74 (10 µM)
1 µl 1:10 dilution of O75 (10 µM)
18 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)


Reaction batch for P217 (Laccase)

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid template (P217 - 1:100 dilution) need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl 1:10 dilution of O74 (10 µM)
1 µl 1:10 dilution of O75 (10 µM)
18 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)

PCR cycling parameters - P251 (Laccase) and P163 (NPT-Cassette)

Segment Cycles Temperature Time
1 1 95 °C 30 s
2 19 95 °C 30 s
52 °C 1 min
68 °C 4 min
4 °C hold
  • After the PCR, the Quickchange product was stored in the fridge.

PCR of AlcR (P61, pAutoRex8) and IRES (P61, pAutoRex8) and analytical gel electrophoresis

Investigator: Louise, Johanna, Christopher

Aim of the experiment: PCR of AlcR (P61, pAutoRex8) and IRES (P61, pAutoRex8).

Procedure:

Operational sequence:

  • PCR reaction mixture for AlcR
volume reagent
10 µl 5x OneTaq Standard Reaction Buffer
1 µl 10 mM dNTPs
1 µl 10 µM Forward Primer (O78 (AlcR_fw))
1 µl 10 µM Reverse Primer (O79 (AlcR_rv))
0.25 µL OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl Plasmid DNA (P61)
35.75 µL ddH2O Water
=50 µL TOTAL
  • PCR reaction mixture for IRES
volume reagent
10 µl 5x OneTaq Standard Reaction Buffer
1 µl 10 mM dNTPs
1 µl 10 µM Forward Primer (O68 (IRES_fw))
1 µl 10 µM Reverse Primer (O69 (IRES_rv))
0.25 µL OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl Plasmid DNA (P61)
35.75 µL ddH2O Water
=50 µL TOTAL
  • Mix with pipette
  • The gradient PCR program was performed after following scheme with following conditions (Tm=56 °C; ΔG=4 °C; AlcR in row 11(=60 °C); IRES in row 1 (=52.0 °C)):
Initial denaturation 94 °C 30 s
30 cycles 94 °C 30 s
Tm=56 °C; ΔG=4 °C 60 s
68 °C 160 s
Final extension 68 °C 5 min
Hold 4 °C infinite
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
  • Resulting products was labeled as F99 (AlcR) and F100 (IRES).

Analytical gelelectrophoresis of F99 (AlcR) and F100 (IRES)

Investigator: Louise, Christopher

Aim of the experiment: Analytical gelelectrophoresis of F99 (AlcR) and F100 (IRES).

Procedure:

  • 4 µl of F99 and F100 were mixed with 0.44  µl DNA loading buffer (10x)
  • Analytical gelelectrophoresis was performed at 90 V for 60 min on a 1% agarose gel.

TUM13 20130613 F100 F99.png

1 kbp DNA ladder F100 F99
as expected as expected

Miniprep of overnight cultures of transformated E. coli XL1 blue with P157, P208, P213, F59+F81(Thermonuclease), F59+F83 (Laccase)

Investigator: Rosario

Aim of the experiment:Miniprep of overnight cultures of transformated E. coli XL1 blue with P157, P208, P213, F59+F81(Thermonuclease), F59+F83 (Laccase)

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
  • The resulting DNA were eluted in tubes labeled as follows:
Content New Tube
P157P245
P208P246
P213P247
F59+F81 IP248
F59+F81 IIP249
F59+F83 IP250
F59+F83 IIP251

Analytical digestion and gelelectrophoresis of P248-P249 (Thermonuclease) and P250-251 (Laccase) with EcoRI and SpeI

Investigator: Rosario

Aim of the experiment: Analytical digestion and gelelectrophoresis of P248-P249 (Thermonuclease) and P250-251 (LAccase) with EcoRI and SpeI

Procedure:

  • Batch
volume reagent
2 µl CutSmart Buffer (10x)
0.25 µl EcoRI
0.25 µl SpeI
15 µl ddH2O
2.5 µl Plasmid DNA (P248-P251)
=20 µl TOTAL
  • Incubation for 60 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min
  • For P248-P251 a 1% agarose gel was used.

TUM13 20130613 anal Verd P248,249,250,251 EcoRI.SpeI.png

Lane: DNA marker 1kb Digestion of P248 with EcoRI & SpeI Digestion of P249 with EcoRI & SpeI Digestion of P250 with EcoRI & SpeI empty Digestion of P251 with EcoRI & SpeI
Result: - as expected as expected no insert (Laccase) - no insert (Laccase)


Transformation of E. coli XL1 blue with P204

Investigator: Johanna

Aim of the experiment: Transformation of E. coli XL1 blue with P204

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 2 µl of DNA was added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • Centrifugation for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated on new chloramphenicol plates.

Sequencing of P61 (pSH21/pAutoRex8)

Investigator: Rosario, Katrin

Aim of the study: Sequencing of P61 (pSH21/pAutoRex8)

Procedure:

Eurofins SmartSeq Kit protocol (15 µl plasmid DNA (50 - 100 ng), 2 µl sequencing primer).

# P61
Name pSH 21 (AlcR/IRES)
O88 (primer 1) FR02305828
O89 (primer 2) FR02305829


Picking of F92+F89, F92+F87, F92+F86, F92+F88, F92+F97, F90+F97, F91+F94, F93+F79, P126 (N-TEV), P127 (C-TEV), P222 (PIF6), P240 (PhyB+Linker) and P244 (IgKappa), F42+F84, F90+F86, F90+F87, F90+F88, F90+F89, F59+F85

Investigator: Rosario, Katrin

Aim of the study: Picking of F92+F89, F92+F87, F92+F86, F92+F88, F92+F97, F90+F97, F91+F94, F93+F79, P126 (N-TEV), P127 (C-TEV), P222 (PIF6), P240 (PhyB+Linker) and P244 (IgKappa), F42+F84, F90+F86, F90+F87, F90+F88, F90+F89, F59+F85

Operational sequence:

  • Picking and overnight culture after standard laboratory's protocol. (4 µl CamR and 4 ml LB-medium)
  • There were no colonies on plates F42+F84 and xy
  • There were a lot of colonies on NK 90 but there were significantly more colonies on the plates with ligation products
  • 2 colonies were picked for every ligation product.

Friday, June 14th

Picking of transformed Plasmid E. coli XL1 blue with P204, F92+F86 and F42+F84

Investigator: Johanna

Aim of the study: Picking of P204, F92+F86 and F42+F84

Operational sequence:

  • Picking and overnight culture after standard laboratory's protocol. (4 µl CamR, 4 ml LB-medium)
  • 3 colonies were picked for every transformation product.

Mass of Minipreps of overnight cultures of transformated E. coli XL1 blue

Investigator: Rosario, Johanna, Andi

Aim of the experiment:Mass of Minipreps of overnight cultures of transformated E. coli XL1 blue.

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
  • The resulting DNA were eluted in tubes labeled as follows:
Content New Tube
F59+F85P252
F59+F85P253
F90+F86P254
F90+F86P255
F90+F87P256
F90+F87P257
F90+F88P258
F90+F88P259
F90+F89P260
F90+F89P261
F90+F97P262
F90+F97P263
F91+F94P264
F91+F94P265
F92+F87P266
F92+F87P267
F92+F97P268
F92+F97P269
F92+F88P270
F92+F88P271
F92+F89P272
F92+F89P273
F93+F79P274
F93+F79P275
P222P276
P222P277
P240P278
P240P279
P126P280
P126P281
P244P282
P244P283
P127P284
P127P285

Transformation of E. coli XL1 blue with P250, P251, P219 (Laccase), P163 (NPT-Cassette)

Investigator: Rosario

Aim of the experiment: Transformation of E. coli XL1 blue with P250, P251, P219 (Laccase), P163 (NPT-Cassette)

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 2 µl of DNA was added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • Centrifugation for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated on new chloramphenicol plates.

Sequencing of P276 (Retrafo of P222, fw), P282 (Retrafo of P244, fw), P278 (Retrafo of P240, fw+rv) and P217 (fw+rv)

Investigators: Louise

Aim of the experiment: Sequencing of P276(Retrafo of P222, fw), P282(Retrafo of P244, fw), P278 (Retrafo of P240, fw+rv) and P217 (fw+rv).

Procedure: Peparation of DNA for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng/µl) and 2 µl sequencing primer 10 µM).


The different genes received the following barcodes:

Label Name Barcode1 Barcode2
P276 PIF6 FR02305830 (O3) -
P282 IgKappa-SigP in pSB1C3 FR02305831 (O3)
P278 PhyB-36AALinker in pSB1C3 FR02305832 (O3) FR02305833 (O4)
P217 Laccase QCIV FR02305826 (O3) FR02305827 (O4)

Analytical digestion and gelelectrophoresis of P252-P275 with EcoRI and SpeI

Investigator: Rosario, Johanna, Louise

Aim of the experiment: Analytical digestion and gelelectrophoresis of P252-P275 with EcoRI and SpeI

Procedure:

  • Batches for every Plasmid of P252-P275 contained
volume reagent
2 µl CutSmart Buffer (10x)
0.25 µl EcoRI
0.25 µl SpeI
15 µl ddH2O
2.5 µl Plasmid DNA (P252-P275)
=20 µl TOTAL
  • The batches for the Quickchangeproduct were prepared with 6 µl of Quickchangeproduct and 2 µl DNA loading buffer.
  • Incubation for 60 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min
  • For P252-P275 a 1% agarose gel was used.

TUM13 20130614 anal Verdau P252-P258 EcoRI.SpeI.png

Lane: DNA marker 1kb Digestion of P252 with EcoRI & SpeI Digestion of P253 with EcoRI & SpeI Digestion of P254 with EcoRI & SpeI Digestion of P255 with EcoRI & SpeI Digestion of P256 with EcoRI & SpeI Digestion of P257 with EcoRI & SpeI Digestion of P258 with EcoRI & SpeI
Result: - corrupt as expected corrupt corrupt corrupt as expected corrupt

TUM13 20130614 anal Verdau P259-P265 EcoRI.SpeI.png

Lane: DNA marker 1kb Digestion of P259 with EcoRI & SpeI Digestion of P260 with EcoRI & SpeI Digestion of P261 with EcoRI & SpeI Digestion of P262 with EcoRI & SpeI Digestion of P263 with EcoRI & SpeI Digestion of P264 with EcoRI & SpeI Digestion of P265 with EcoRI & SpeI
Result: - as expected as expected (sequencing necessary) as expected (sequencing necessary) bad miniprep as expected not sure (has to be repeated with control P278) not sure (has to be repeated with control P278)

TUM13 20130614 anal Verdau P266-P272 EcoRI.SpeI.png

Lane: DNA marker 1kb Digestion of P266 with EcoRI & SpeI Digestion of P267 with EcoRI & SpeI Digestion of P268 with EcoRI & SpeI Digestion of P269 with EcoRI & SpeI Digestion of P270 with EcoRI & SpeI Digestion of P271 with EcoRI & SpeI Digestion of P272 with EcoRI & SpeI
Result: - as expected as expected as expected as expected as expected as expected as expected (has to be sequenced)

TUM13 20130614 anal Verdau P273-P275 P251 P163I P163II P217 EcoRI.SpeI 2.png TUM13 20130614 anal Verdau P273-P275 P251 P163I P163II P217 EcoRI.SpeI.png

Lane: DNA marker 1kb Digestion of P273 with EcoRI & SpeI Digestion of P274 with EcoRI & SpeI Digestion of P275 with EcoRI & SpeI Digestion of QC I product of P251 with DpnI Digestion of QC I product of P163I with DpnI Digestion of QC V product of P163II with DpnI Digestion of QC V product of P217 with DpnI
Result: - as expected (has to be sequenced) as expected (has to be sequnced) as expected (has to be sequnced) corrupt, no QC product corrupt, no QC product corrupt, no QC product as expected, QC product


Preparative Digestion of IRES (F100) and AlcR (F99) with EcoRI and SpeI

volume reagent
4 µl CutSmart Buffer (10x)
1 µl EcoRI
1 µl SpeI
9 µl ddH2O
25 µl Fragment DNA (F99 or F100)
=40 µl TOTAL
  • Incubation for 150 min at 37 °C
  • Purification by QIAGEN PCR Purification Kit
  • new Names: F99 -> F101 and F100 -> F102

Oligohybridization of Strep-TEV (O90 + O91)

Investigator: Rosario

Aim of the experiment: Oligohybridization of Strep-TEV (O90 + O91)

Procedure:

  • 25 µL of 100 pmol/µl of Strep-TEV_fw and 25 µL of 100 pmol/µl Strep-TEV_rev
  • Heating up to 95 °C for 5 min
  • Cooling at RT in a styropor box overnight

Saturday, June 15th

Miniprep of overnight cultures of transformated E. coli XL1 blue with P204 (IgKappa-SigP in pSB1C3), F92+F82 (IgKappa-SigP_EreB in pSB1C3)

Investigator: Jeff

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with P204 (IgKappa-SigP in pSB1C3), F92+F82 (IgKappa-SigP_EreB in pSB1C3).

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
  • The resulting DNA were eluted in tubes labeled as follows:
Content New Tube Concentration in ng/µl
ReTraFo of P204 (IgKappa-SigP in pSB1C3)P286105.7
ReTraFo of P204 (IgKappa-SigP in pSB1C3)P287303.6
ReTraFo of P204 (IgKappa-SigP in pSB1C3)P288268.1
F92+F86 (IgKappa-SigP_EreB in pSB1C3)P289121.2
F92+F86 (IgKappa-SigP_EreB in pSB1C3)P290402.3
F92+F86 (IgKappa-SigP_EreB in pSB1C3)P291405.2

Ligation of F41+F101 (AlcR) and F41+F102 (IRES of poliovirus)

Investigator: Jeff

Aim of the experiment: Ligation of F41+F101 (AlcR) and F41+F102 (IRES of poliovirus).

Procedure:

  • Ligation batch for F41+F101 (AlcR), 50 ng vector used, 1:1:
volume reagent
0.56 µl F41 (89.9 ng/µl, 2086 bp)
5.23 µl F101 (11.3 ng/µl, 2466 bp)
11.21 µl ddH2O
2 µl T4 ligase buffer (10x)
0.5 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F41+F102 (IRES of poliovirus), 100 ng vector used, 3:1:
volume reagent
1.11 µl F41 (89.9 ng/µl, 2086 bp)
7.92 µl F102 (11.4 ng/µl, 628 bp)
7.97 µl ddH2O
2 µl T4 ligase buffer (10x)
0.5 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
  • The ligation was performed for 1 hour at room temperature.

Analytical digestion and gelelectrophoresis of P289-P291 (IgKappa-SigP_EreB in pSB1C3) with EcoRI & SpeI

Investigator: Jeff

Aim of the experiment: Analytical digestion and gelelectrophoresis of P289-P291 (IgKappa-SigP_EreB in pSB1C3) with EcoRI & SpeI.

Procedure:

  • Batch
volume reagent
2 µl CutSmart Buffer (10x)
0.25 µl EcoRI-HF
0.25 µl SpeI-HF
15 µl ddH2O
2.5 µl Plasmid DNA (P289-P291, P245)
=20 µl TOTAL
  • Incubation for 60 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min on a 1% agarose gel.

TUM13 20130615 anal gel P289-P291 EcoRI.SpeI P245 EcoRI.SpeI.png

Lane: DNA ladder 100bp Digestion of P289 (IgKappa-SigP_EreB in pSB1C3) with EcoRI & SpeI, clone 1 Digestion of P290 (IgKappa-SigP_EreB in pSB1C3) with EcoRI & SpeI, clone 2 Digestion of P291 (IgKappa-SigP_EreB in pSB1C3) with EcoRI & SpeI, clone 3 Digestion of P245 (EreB in pSB1C3) with EcoRI & SpeI
Result: as expected as expected as expected as expected (control)

Transformation of E. coli XL1 blue with F41+F101 (AlcR in pSB1C3) and F41+F102 (IRES in pSB1C3)

Investigator: Jeff

Aim of the experiment: Transformation of E. coli XL1 blue with F41+F101 (AlcR in pSB1C3) and F41+F102 (IRES in pSB1C3).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of ligation product was added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Sunday, June 16th

QuikChange mutagenesis to remove forbidden restriction sites - SDMIV - of P217 (Laccase), P163 (NPT-Cassette)

Investigator: Andi

Aim of the experiment: Removal of forbidden restriction site from P217 (Laccase) and P163 (NPT-Cassette).

Procedure: Quikchange - PCR

Reaction batch for P217 (Laccase)

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid template (P217 - 1:100 dilution) need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl 1:10 dilution of O74 (10 µM)
1 µl 1:10 dilution of O75 (10 µM)
18 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)

Reaction batch for P163 (NPT-Cassette)

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid template (P163 - 1:50 dilution) need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl 1:10 dilution of O70 (10 µM)
1 µl 1:10 dilution of O71 (10 µM)
18 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)

PCR cycling parameters - P251 (Laccase) and P163 (NPT-Cassette)

Segment Cycles Temperature Time
1 1 95 °C 30 s
2 19 95 °C 30 s
52 °C 1 min
68 °C 5 min
4 °C hold
  • After the PCR, the Quickchange product was stored in the fridge.

Picking of transformed Plasmid E. coli XL1 blue with QC of P217, P163, F41+F101 and F41+F102

Investigator: Andi, Jeff

Aim of the study: Picking of QC of P217, P163, F41+F101 and F41+F102

Operational sequence:

  • Picking and overnight culture after standard laboratory's protocol. (4 µl Cam, 4 ml LB-medium)
  • 1 colonies were picked for every transformation product.

Ligation of F59+F74 (SERK-SigP) and F59+F103 (Strep-TEV)

Investigator: Andi, Jeff

Aim of the experiment: Ligation of F59+F74 (SERK-SigP) and F59+F103 (Strep-TEV).

Procedure:

  • Ligation batch for F59+F74 (SERK-SigP), 100 ng vector used, insert:vector = 3:1
volume reagent
1.5 µl F59 (66.7 ng/µl, 2086 bp)
3 µl F74 (4.8 ng/µl, 100 bp)
12.5 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F59+F103 (Strep-TEV), 100 ng vector used, insert:vector = 3:1
volume reagent
1.5 µl F59 (66.7 ng/µl, 2086 bp)
15.5 µl F103 (504.9 ng/µl, 54 bp)
7.97 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Negative control was prepared by replacing the volume of the insert by ddH2O.
  • The ligation was performed for 1 hour at room temperature.


Transformation of E. coli XL1 blue with P217 (QC of 16.05.2013), P163 (QC of 16.05.2013), P7 (Retrafo of PhyB) and ligation products F59+F74 (SERK-SigP in pSB1C3) and F59+F103 (Strep-TEV in pSB1C3)

Investigator: Andi, Jeff

Aim of the experiment: Transformation of E. coli XL1 blue with P217 (QC of 16.05.2013) and P163 (QC of 16.05.2013), P7 (Retrafo of PhyB) and ligation products F59+F74 (SERK-SigP in pSB1C3) and F59+F103 (Strep-TEV in pSB1C3).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of ligation product/ 2 µl of plasmid (P7) was added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Week 9

Monday, June 17th

Miniprep of overnight cultures of transformated E. coli XL1 blue with QC I of P163 (NPT-casette), QC V of P217 (laccase), ligation products F41+F101 (AlcR) and F41+F102 (IRES)

Investigator: Rosario

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with QC I of P163 (NPT-casette), QC V of P217 (laccase), ligation products F41+F101 (AlcR) and F41+F102 (IRES) Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
  • The resulting DNA were eluted in tubes labeled as follows:
Content New Tube
QC I of P163P292
QC V of P217P293
F41+F101 IP294
F41+F101 IIP295
F41+F101 IIIP296
F41+F102 IP297
F41+F102 IIP298
F41+F102 IIIP299

Picking of transformed Plasmid E. coli XL1 blue with P217 (QC of 16.05.2013), P163 (QC of 16.05.2013), P7 (Retrafo of PhyB) and ligation products F59+F74 (SERK-SigP in pSB1C3) and F59+F103 (Strep-TEV in pSB1C3)

Investigator: Rosario

Aim of the study: Picking of transformed Plasmid E. coli XL1 blue with P217 (QC of 16.05.2013), P163 (QC of 16.05.2013), P7 (Retrafo of PhyB) and ligation products F59+F74 (SERK-SigP in pSB1C3) and F59+F103 (Strep-TEV in pSB1C3)

Operational sequence:

  • Picking and overnight culture after standard laboratory's protocol. (4 µl Cam, 4 ml LB-medium)
  • 1 colony was picked for every transformation product.

Preparative digestion and gelelectrophoresis of P264 (PhyBlinker+cTev) with AgeI/SpeI, P299 (IRES) with NgoMIV/SpeI, P274 (nTevlinker)with AgeI/SpeI, P111 (TEV) with AgeI/SpeI and P248 (Nuclease) with NgoMIV/SpeI

Investigator: Johanna, Flo

Aim of the experiment: Preparative digestion and gelelectrophoresis of P264 (PhyBlinker+cTev) with AgeI/SpeI, P299 (IRES) with NgoMIV/SpeI, P274 (nTevlinker)with AgeI/SpeI, P111 (TEV) with AgeI/SpeI and P248 (Nuclease) with NgoMIV/SpeI

Procedure:

  • Batch for preparative digestion of P264 (PhyBlinker+cTev) with AgeI/SpeI
volume reagent
20 µl P264
4 µl CutSmart Buffer
1 µl AgeI(20 U/µl)
1 µl SpeI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P299 (IRES) with NgoMIV/SpeI
volume reagent
20 µl P299
4 µl CutSmart Buffer
1 µl NgoMIV(20 U/µl)
1 µl SpeI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P274 (nTevlinker)with AgeI/SpeI
volume reagent
20 µl P274
4 µl CutSmart Buffer
1 µl AgeI(20 U/µl)
1 µl SpeI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P111 (TEV) with AgeI/SpeI
volume reagent
20 µl P111
4 µl CutSmart Buffer
1 µl AgeI(20 U/µl)
1 µl SpeI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P248 (Nuclease) with NgoMIV/SpeI
volume reagent
20 µl P248
4 µl CutSmart Buffer
1 µl NgoMIV(20 U/µl)
1 µl SpeI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Incubation for 2.5 h at 37 °C.
  • 4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and were loaded on a 1% agarose gel for preparative gelelectrophoresis.
  • Preparative gelelectrophoresis was performed at 90 V for 60 min.

TUM13 20130617 prep Verd P111 AgeI.SpeI P248 NgoMIV.SpeI P264 AgeI.SpeI.png

Lane: Digestion of P111 with AgeI & SpeI 1 kbp DNA ladder Digestion of P248 with NgoMIV & SpeI Digestion of P264 with AgeI & SpeI
Result: as expected as expected as expected

TUM13 20130617 prep Verd P274 AgeI.SpeI P299 NgoMIV.SpeI.png

Lane: Digestion of P274 with AgeI & SpeI 1 kbp DNA ladder Digestion of P299 with NgoMIV & SpeI
Result: low concentration, discarded not digested, discarded
  • The DNA was extracted from the gel with QIAquick Gel Extraction Kit, QIAGEN

Analytical digestion and gelelectrophoresis of P293-P299 with EcoRI & SpeI and P292 with EcoRI & PstI

Investigator: Rosario

Aim of the experiment: Analytical digestion and gelelectrophoresis of P293-P299 with EcoRI & SpeI and P292 with EcoRI & PstI Procedure:

  • Batch
volume reagent
2 µl CutSmart Buffer (10x)
0.25 µl EcoRI-HF
0.25 µl SpeI-HF (PstI-HF for P292)
15 µl ddH2O
2.5 µl Plasmid DNA (P292-P299)
=20 µl TOTAL
  • Incubation for 60 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min on a 1% agarose gel.

TUM13 20130617 anal Verdau P292 EcoRI.SpeI P293 EcoRI.PstI P294-P299 EcoRI.SpeI 2.png

Lane: 1 kbp DNA ladder Digestion of P292 (QC I P163 (NPT-casette)) with EcoRI & PstI Digestion of P293 (QC V P217 (Laccase)) with EcoRI & SpeI Digestion of P294 (F41+F101 (AlcR)) with EcoRI & SpeI, clone 1 Digestion of P295 (F41+F101 (AlcR)) with EcoRI & SpeI, clone 2 Digestion of P296 (F41+F101 (AlcR)) with EcoRI & SpeI, clone 3 Digestion of P297 (F41+F102 (IRES)) with EcoRI & SpeI, clone 1 Digestion of P298 (F41+F102 (IRES)) with EcoRI & SpeI, clone 2 Digestion of P299 (F41+F102 (IRES)) with EcoRI & SpeI, clone 3 1 kbp DNA ladder
Result: corrupt corrupt as expected as expected as expected as expected as expected as expected

DpnI digestion of P163QCI and P217QCV

Investigator: Flo, Jeff

Aim of the experiment: DpnI digestion of P163QCI and P217QCV.

Procedure:

  • 1 µl of DpnI was added to the QuikChange products of P163QCI and P217QCV.
  • The mixtures were incubated for 1 h at 37 °C; afterwards they were ready for transformation.

Analytical gelelectrophoresis of DpnI digestion of P163QCI and P217QCV

Investigator: Jeff, Flo

Aim of the experiment: Analysis of the DpnI digestion of P163QCI and P217QCV

Procedure:

  • 4.5 µl of the QuikChange products of P163 and P217, named P163QCI and P217QCV were mixed together with 0.5 µl of DNA loading buffer (10x).
  • Analytical gelelectrophoresis was performed at 90 V for 30 min.

TUM13 20130617 QC P163QCI.1 P163QCI.2 P217QCV.1 P217QCV.2.png

Lane: 1 kbp DNA ladder DpnI digestion of P163QCI.1 DpnI digestion of P163QCI.2 DpnI digestion of P217QCV.1 DpnI digestion of P217QCV.2
Result: empty empty empty empty


Ligation of F77+F105 (NLS in pSB1C3 + mature thermonuclease)

Investigator: Jeff, Flo

Aim of the experiment: Ligation of F77+F105 (NLS in pSB1C3 + mature thermonuclease).

Procedure:

  • Ligation batch for F77+F105 (NLS in pSB1C3 + mature thermonuclease), 100 ng vector used, insert:vector = 3:1
volume reagent
1.78 µl F77 (56.2 ng/µl, 2143 bp)
5.22 µl F105 (12 ng/µl, 447 bp)
10 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Negative control was prepared by replacing the volume of the insert by ddH2O.
  • The ligation was performed overnight at 16 °C.

Transformation of E. coli XL1 blue with P217 (QC of 16.05.2013), P163 (QC of 16.05.2013) and P160, P264, P274 and P275 for retransformations

Investigator: Flo

Aim of the experiment: Transformation of E. coli XL1 blue with P217 (QC of 16.05.2013), P163 (QC of 16.05.2013) and P160, P264, P274 and P275 for retransformations.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of QuikChange product/2 µl of plasmid DNA was added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.


Sequencing of P248 (Nuc A, fw), P275 (N-TEV_36AAlinker, fw), P299 (IRES, fw), P292 (QC I of P163 NPT-casette,fw+rv), P293 (QC V of P217 Laccase,fw+rv) and P290 (IgKappa_EreB,fw+rv)

Investigators: Rosario

Aim of the experiment: Sequencing of P248 (Nuc A, fw), P275 (N-TEV_36AAlinker, fw), P299 (IRES, fw), P292 (QC I of P163 NPT-casette,fw+rv), P293 (QC V of P217 Laccase,fw+rv) and P290 (IgKappa_EreB,fw+rv).

Procedure: Peparation of DNA for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng/µl) and 2 µl sequencing primer 10 µM).


The different genes received the following barcodes:

Label Name Barcode1 Barcode2
P248 NucA FR02305834 (O3) -
P275 N-TEV_36AAlinker FR02305835 (O3)
P299 IRES FR02305825 (O3) -
P292 QC I NPT-casette FR02305824 (O3) FR02305823 (O4)
P293 QC V Laccase FR02305822 (O3) FR02305821 (O4)
P290 IgKappa_EreB FR02305820 (O3) FR02305819 (O4)

Tuesday, June 18th

Picking of E. coli XL1 blue transformated with P160, P264, P274 and P275

Investigator: Rosario

Aim of the study: Picking of E. coli XL1 blue transformated with P160, P264, P274 and P275.

Operational sequence:

  • Picking and overnight culture after standard laboratory's protocol. (4 µl Cam, 4 ml LB-medium)
  • 2 colonies were picked for every transformation product.

Transformation of E. coli XL1 blue with F77+F105 (NLS in pSB1C3 + mature thermonuclease) and P299 Retrafo

Investigator: Rosario

Aim of the experiment: Transformation of E. coli XL1 blue with P217 (QC of 16.05.2013), P163 (QC of 16.05.2013) and P160, P264, P274 and P275 for retransformations.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 2 µl of plasmid DNA was added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Miniprep of overnight cultures of transformated E. coli XL1 blue with F59+F103 (Strep-TEV-Linker in PsB1C3) and F59+F74 (Serk-Sigp in PsB1C3) and P7 (PhyB)

Investigator: Louise

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with F59+F103 (Strep-TEV-Linker in PsB1C3) and F59+F74 (Serk-Sigp in PsB1C3) and P7 (PhyB).

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
  • The resulting DNA were eluted in tubes labeled as follows:
Content New Tube
P7 (PhyB) clone 1P300
P7 (PhyB) clone 2P301
F59+F74 (Serk-Sigp in PsB1C3) clone 1P302
F59+F74 (Serk-Sigp in PsB1C3) clone 2P303
F59+F103 (Strep-TEV-Linker in PsB1C3) clone 1P304
F59+F103 (Strep-TEV-Linker in PsB1C3) clone 2P305

Analytical digestion and gelelectrophoresis of P300-P301 (PhyB), P302-P303 (Serk-Sigp in PsB1C3) and P304-P305 (Strep-TEV-Linker in PsB1C3) with XbaI and AgeI

Investigator: Louise, Rosario, Florian

Aim of the experiment: Analytical digestion and gelelectrophoresis of P300-P301 (PhyB), P302-P303 (Serk-Sigp in PsB1C3) and P304-P305 (Strep-TEV-Linker in PsB1C3) with XbaI and AgeI.

Procedure:

  • Batch
volume reagent
2 µl CutSmart Buffer (10x)
0.25 µl AgeI
0.25 µl XbaI
15 µl ddH2O
2.5 µl Plasmid DNA (P300-P305)
=20 µl TOTAL
  • Incubation for 60 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min

TUM13 20130618 anal Verdau P300-P305 EcoRI.PstI.png

Lane: Digestion of P300 with XbaI/AgeI Digestion of P301 with XbaI/AgeI Digestion of P302 with XbaI/AgeI Digestion of P303 with XbaI/AgeI Digestion of P304 with XbaI/AgeI Digestion of P305 with XbaI/AgeI 1 kb DNA Ladder 100 bp DNA Ladder
Result: as expected as expected as expected as expected as expected as expected

Preparative digestion and gelelectrophoresis of P212 (SERK-TMD) with NgoMIV/SpeI, P238 (SERK-TMD_Linker_GFP) with NgoMIV/SpeI, P303 (SERK-SigP) with AgeI/SpeI and P304 (StrepTEV-Linker) with AgeI/SpeI

Investigator: Louise, Flo, Jeff, Rosario

Aim of the experiment: Preparative digestion and gelelectrophoresis of P212 (SERK-TMD) with NgoMIV/SpeI, P238 (SERK-TMD_Linker_GFP) with NgoMIV/SpeI, P303 (SERK-SigP) with AgeI/SpeI and P304 (StrepTEV-Linker) with AgeI/SpeI.

Procedure:

  • Batch for preparative digestion of P212 (SERK-TMD) with NgoMIV/SpeI
volume reagent
20 µl P212
4 µl CutSmart Buffer
1 µl NgoMIV(20 U/µl)
1 µl SpeI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P238 (SERK-TMD_Linker_GFP) with NgoMIV/SpeI
volume reagent
20 µl P238
4 µl CutSmart Buffer
1 µl NgoMIV(20 U/µl)
1 µl SpeI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P303 (SERK-SigP) with AgeI/SpeI
volume reagent
20 µl P303
4 µl CutSmart Buffer
1 µl AgeI(20 U/µl)
1 µl SpeI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P304 (StrepTEV-Linker) with AgeI/SpeI
volume reagent
20 µl P304
4 µl CutSmart Buffer
1 µl AgeI(20 U/µl)
1 µl SpeI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Incubation for 2.5 h at 37 °C.
  • 4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and P238, P303 and P304 were loaded on a 1% agarose gel, while P212 was loaded on a 2% agarose gel for preparative gelelectrophoresis.
  • Preparative gelelectrophoresis was performed at 90 V for 60 min.

TUM13 20130618 prep Verd P238 NgoMIV.AgeI P303 AgeI.SpeI P304 AgeI.SpeI.png

Lane: Digestion of P238 (SERK-TMD_Linker_GFP) with NgoMIV/SpeI 1 kbp DNA ladder Digestion of P303 (SERK-SigP) with AgeI/SpeI Digestion of P304 (StrepTEV-Linker) with AgeI/SpeI
Result: as expected as expected as expected

TUM13 20130618 prep Verd P212 NgoMIV.SpeI.png

Lane: 1 kbp DNA ladder Digestion of P212 (SERK-TMD) with NgoMIV/SpeI 100 bp DNA Ladder
Result: as expected
  • The DNA was extracted from the gel with QIAquick Gel Extraction Kit, QIAGEN

Ligation of F109+F86 (SERK-SigP + EreB), F109+F87 (SERK-SigP + NanoLuc), F109+F88 (SERK-SigP + SpyCatcher), F109+F89 (SERK-SigP + SpyTag), F109+F97 (SERK-SigP + XylE), F109+F107 (SERK-SigP + SERK-TMD) and F110+F108 (Strep-tag-TEV-site-linker + SERK-TMD_8AAlinker_GFPmut1)

Investigator: Jeff, Flo

Aim of the experiment: Ligation of F109+F86 (SERK-SigP + EreB), F109+F87 (SERK-SigP + NanoLuc), F109+F88 (SERK-SigP + SpyCatcher), F109+F89 (SERK-SigP + SpyTag), F109+F97 (SERK-SigP + XylE), F109+F107 (SERK-SigP + SERK-TMD) and F110+F108 (Strep-tag-TEV-site-linker + SERK-TMD_8AAlinker_GFPmut1).

Procedure:

  • Ligation batch for F109+F86 (SERK-SigP + EreB), 100 ng vector used, insert:vector = 3:1
volume reagent
0.92 µl F109 (108.2 ng/µl, 2177 bp)
5.57 µl F86 (31.0 ng/µl, 1254 bp)
10.51 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F109+F87 (SERK-SigP + NanoLuc), 100 ng vector used, insert:vector = 3:1
volume reagent
0.92 µl F109 (108.2 ng/µl, 2177 bp)
6.89 µl F87 (10.2 ng/µl, 510 bp)
9.19 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F109+F88 (SERK-SigP + SpyCatcher), 100 ng vector used, insert:vector = 3:1
volume reagent
0.92 µl F109 (108.2 ng/µl, 2177 bp)
5.37 µl F88 (8.7 ng/µl, 339 bp)
10.71 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F109+F89 (SERK-SigP + SpyTag), 100 ng vector used, insert:vector = 3:1
volume reagent
0.92 µl F109 (108.2 ng/µl, 2177 bp)
12.0 µl F89 (6.8 ng/µl, 39 bp)
4.08 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F109+F97 (SERK-SigP + XylE), 100 ng vector used, insert:vector = 3:1
volume reagent
0.92 µl F109 (108.2 ng/µl, 2177 bp)
6.52 µl F97 (19.4 ng/µl, 918 bp)
9.56 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F109+F107 (SERK-SigP + SERK-TMD), 100 ng vector used, insert:vector = 3:1
volume reagent
0.92 µl F109 (108.2 ng/µl, 2177 bp)
5.38 µl F107 (4.8 ng/µl, 186 bp)
10.70 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F110+F108 (Strep-tag-TEV-site-linker + SERK-TMD_8AAlinker_GFPmut1), 100 ng vector used, insert:vector = 3:1
volume reagent
1.38 µl F110 (72.4 ng/µl, 2143 bp)
5.53 µl F108 (23.6 ng/µl, 933 bp)
10.09 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Negative control was prepared by replacing the volume of the insert by ddH2O.
  • The ligation was performed for 1 hour at room temperature.

QuikChange mutagenesis to remove forbidden restriction sites - SDMV of P217 (Laccase RFC10) and SDMI of P162 (NPT-Cassette RFC10)

Investigator: Jeff

Aim of the experiment: Removal of the last existing forbidden restriction site from P217 (Laccase) and the first one from P162 (NPT-Cassette).

Procedure: Quikchange - PCR

Reaction batch for P217 (Laccase)

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
0.5 µl Plasmid template (P217 - 1:100 dilution) need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl 1:10 dilution of O74 (=10 µM)
1 µl 1:10 dilution of O75 (=10 µM)
18.5 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)

Reaction batch for P163 (NPT-Cassette)

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
0.5 µl Plasmid template (P162 - 1:50 dilution) need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl 1:10 dilution of O70 (10 µM)
1 µl 1:10 dilution of O71 (10 µM)
18.5 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)

PCR cycling parameters - P251 (Laccase) and P163 (NPT-Cassette)

Segment Cycles Temperature Time
1 1 95 °C 30 s
2 19 95 °C 30 s
52 °C 1 min
68 °C 4 min
4 °C hold
  • After the PCR, the Quickchange products were mixed with 1 µl of DpnI (10 U/µl) and incubated at 37 °C for 1 h.

Analytical gelelectrophoresis of the DpnI digested products of the performed QuikChange of SDMV of P217 (Laccase RFC10) and SDMI of P162 (NPT-Cassette RFC10)

Investigator: Jeff

Aim of the experiment:

Procedure:

  • 4.5 µl of each of the DpnI digested QuikChange products were mixed with 1 µl of DNA loading buffer (10x).
  • Analytical gelelectrophoresis was performed at 90 V for 40 min at an 1% agarose gel.

TUM13 20130618 QC P162QCI P217QCV.png

Lane: 1 kbp DNA ladder DpnI digestion of P163QCI DpnI digestion of P217QCV
Result: as expected as expected

Transformation of E. coli XL1-blue with ligation products F109+F86 (SERK-SigP + EreB), F109+F87 (SERK-SigP + NanoLuc), F109+F88 (SERK-SigP + SpyCatcher), F109+F89 (SERK-SigP + SpyTag), F109+F97 (SERK-SigP + XylE), F109+F107 (SERK-SigP + SERK-TMD) and F110+F108 (Strep-tag-TEV-site-linker + SERK-TMD_8AAlinker_GFPmut1), with DpnI digested QuikChange products P162QCI and P217QCV, and P212 (ReTraFo), P238 (ReTraFo)

Investigator: Jeff

Aim of the experiment: Transformation of E. coli XL1-blue with ligation products F109+F86 (SERK-SigP + EreB), F109+F87 (SERK-SigP + NanoLuc), F109+F88 (SERK-SigP + SpyCatcher), F109+F89 (SERK-SigP + SpyTag), F109+F97 (SERK-SigP + XylE), F109+F107 (SERK-SigP + SERK-TMD) and F110+F108 (Strep-tag-TEV-site-linker + SERK-TMD_8AAlinker_GFPmut1), with DpnI digested QuikChange products P162QCI and P217QCV, and P212 (ReTraFo), P238 (ReTraFo).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of QuikChange products/10 µl of ligation products/2 µl of plasmid DNA was added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Sequencing of P294 (AlcR)

Investigators: Rosario

Aim of the experiment: Sequencing of P294 (AlcR).

Procedure: Peparation of DNA for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng/µl) and 2 µl sequencing primer 10 µM).


The different genes received the following barcodes:

Label Name Barcode1 Barcode2
P294 AlcR FR02305817 (O3) FR02305818(O4)

Wednesday, June 19th

QuikChange mutagenesis to remove forbidden restriction sites - SDMI of P294 (AlcR)

Investigator: Florian - Master of QuikChange 2.0

Aim of the experiment: Removal of the first forbidden restriction site from P294 (AlcR).

Procedure: Quikchange - PCR

Reaction batch for P294 (AlcR)

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
0.5 µl Plasmid template (P294 - 1:50 dilution) need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl 1:10 dilution of O80 (=10 µM)
1 µl 1:10 dilution of O81 (=10 µM)
18.5 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)

PCR cycling parameters - P294 (AlcR)

Segment Cycles Temperature Time
1 1 95 °C 30 s
2 19 95 °C 30 s
52 °C 1 min
68 °C 5 min
4 °C hold
  • After the PCR, the Quickchange products were mixed with 1 µl of DpnI (10 U/µl) and incubated at 37 °C for 1 h.

Analytical gelelectrophoresis of the DpnI digested products of the performed QuikChange of SDMI of P294 (AlcR)

Investigator: Florian

Aim of the experiment: Analytical gelelectrophoresis of the DpnI digested products of the performed QuikChange of SDMI of P294 (AlcR).

Procedure:

  • 4 µl of each of the DpnI digested QuikChange products were mixed with 1 µl of DNA loading buffer (10x).
  • Analytical gelelectrophoresis was performed at 90 V for 50 min at an 1% agarose gel.

TUM13 20130619 QCI AlcR.png

Lane: 1 kbp DNA ladder DpnI digestion of P294QCI
Result: only Primers

Transformation of E. coli XL1-blue with DpnI digested QuikChange product P294QCI and P294 (ReTrafo of FluA)

Investigator: Florian

Aim of the experiment: Transformation of E. coli XL1-blue with DpnI digested QuikChange product P294QCI and P294 (ReTrafo of FluA).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of QuikChange products/2 µl of plasmid DNA was added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol/ampicillin plate.

Sequencing of P308 (ReTrafo of P264, PhyB_36AALinker_C-TEV in pSB1C3), P310 (ReTrafo of P274, N-TEV_36AALinker in pSB1C3), P303 (SERK-SigP in pSB1C3), P304 (Strep-TEV-Linker in pSB1C3), P305 (Strep-TEV-Linker in pSB1C3) and P301 (ReTrafo of P7, Phytotchrome B)

Investigators: Rosario

Aim of the experiment: Sequencing of P308 (ReTrafo of P264, PhyB_36AALinker_C-TEV in pSB1C3), P310 (ReTrafo of P274, N-TEV_36AALinker in pSB1C3), P303 (SERK-SigP in pSB1C3), P304 (Strep-TEV-Linker in pSB1C3), P305 (Strep-TEV-Linker in pSB1C3) and P301 (ReTrafo of P7, Phytotchrome B).

Procedure: Peparation of DNA for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng/µl) and 2 µl sequencing primer 10 µM).


The different genes received the following barcodes:

Label Name Barcode1 Barcode2
P308 PhyB_36AALinker_C-TEV in pSB1C3 FR02305816 (O3) FR02305815 (O4)
P310 N-TEV_36AALinker in pSB1C3 FR02305814 (O3)
P303 SERK-SigP in pSB1C3 FR02305813 (O3) -
P304 Strep-TEV-Linker in pSB1C3 FR02305812 (O3) -
P305 Strep-TEV-Linker in pSB1C3 FR02305811 (O3) -
P301 Phytotchrome B FR02305810 (O3) FR02305809 (O4)

Miniprep of overnight cultures of transformated E. coli XL1 blue with ReTrafo of P160 (SV40 NLS), ReTrafo of P264 (PhyB_36AALinker_C-TEV in pSB1C3), ReTrafo of P274 (N-TEV_36AALinker in pSB1C3) and ReTrafo of P275 (N-TEV_36AALinker in pSB1C3)

Investigator: Rosario

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with ReTrafo of P160 (SV40 NLS), ReTrafo of P264 (PhyB_36AALinker_C-TEV in pSB1C3), ReTrafo of P274 (N-TEV_36AALinker in pSB1C3) and ReTrafo of P275 (N-TEV_36AALinker in pSB1C3).


Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
  • The resulting DNA were eluted in tubes labeled as follows:
Content New Tube
P160 IP306
P160 IIP307
P264 IP308
P264 IIP309
P274 IP310
P274 IIP311
P275 IP312
P275 IIP313

Pouring of CamR gel plates

Investigator: Louise, Volker

Aim of the experiment: Pouring of CamR gel plates

Procedure: Preparation of 5 l of LB-Medium with agar was performed after standard recipe

Picking of QC I of P162 (npt-casette in pSB1C3), QC V of P217 (Laccase), ReTrafo of P212, ReTrafo of P238, ReTrafo of P299. F77+F105 (NucA+NLS), F110+F108 (Strep-tag-TEV-site-linker+SERK-TMD_8AAlinker_GFPmut1), F109+F86 (SERK-SigP+EreB), F109+F87 (SERK-SigP+NanoLuc), F109+F88 (SERK-SigP+SpyCatcher), F109+F89 (SERK-SigP+SpyTag), F109+F97 (SERK-SigP+xylE), F109+F107 (SERK-SigP+SERK-TMD)

Investigator: Rosario

Aim of the experiment: Picking of QC I of P162 (npt-casette in pSB1C3), QC V of P217 (Laccase), ReTrafo of P212, ReTrafo of P238, ReTrafo of P299. F77+F105 (NucA+NLS), F110+F108 (Strep-tag-TEV-site-linker+SERK-TMD_8AAlinker_GFPmut1), F109+F86 (SERK-SigP+EreB), F109+F87 (SERK-SigP+NanoLuc), F109+F88 (SERK-SigP+SpyCatcher), F109+F89 (SERK-SigP+SpyTag), F109+F97 (SERK-SigP+xylE), F109+F107 (SERK-SigP+SERK-TMD)

Procedure:

  • Retransformations (P212, P238, P299) 1 clone each
  • Quickchanges (QCI P162, QCV P217) 2 clones each
  • Ligation F77+F105 4 clones because of the high number of colonies at F77 negative ctrl
  • All other Ligations (F110+F108, F109+F86, F109+F87, F109+F88, F109+F89, F109+F97, F109+F107) 2 clones each

Thursday, June 20th

Miniprep of overnight cultures of transformated E. coli XL1 blue with QC I of P162 (npt-casette in pSB1C3), QC V of P217 (Laccase), ReTrafo of P212, ReTrafo of P238, ReTrafo of P299. F77+F105 (NucA+NLS), F110+F108 (Strep-tag-TEV-site-linker+SERK-TMD_8AAlinker_GFPmut1), F109+F86 (SERK-SigP+EreB), F109+F87 (SERK-SigP+NanoLuc), F109+F88 (SERK-SigP+SpyCatcher), F109+F89 (SERK-SigP+SpyTag), F109+F97 (SERK-SigP+xylE), F109+F107 (SERK-SigP+SERK-TMD)

Investigator: Rosario

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with QC I of P162 (npt-casette in pSB1C3), QC V of P217 (Laccase), ReTrafo of P212, ReTrafo of P238, ReTrafo of P299. F77+F105 (NucA+NLS), F110+F108 (Strep-tag-TEV-site-linker+SERK-TMD_8AAlinker_GFPmut1), F109+F86 (SERK-SigP+EreB), F109+F87 (SERK-SigP+NanoLuc), F109+F88 (SERK-SigP+SpyCatcher), F109+F89 (SERK-SigP+SpyTag), F109+F97 (SERK-SigP+xylE), F109+F107 (SERK-SigP+SERK-TMD).

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
  • The resulting DNA were eluted in tubes labeled as follows:
Content New Tube
QC I P162 IP315
QC I P162 IIP316
QC V P217 IP317
QC V P217 IIP318
P212 ReTrafoP319
P238 ReTrafoP320
P299 ReTrafoP321
F77+F105 IP322
F77+F105 IIP323
F77+F105 IIIP324
F77+F105 IVP325
F110+F108 IP326
F110+F108 IIP327
F109+F86 IP328
F109+F86 IIP329
F109+F87 IP330
F109+F87 IIP331
F109+F88 IP332
F109+F88 IIP333
F109+F89 IP334
F109+F89 IIP335
F109+F97 IP336
F109+F97 IIP337
F109+F107 IP338
F109+F107 IIP339

Retransformation of E. coli XL1 blue with P170, P111, P314, P160, P275, P244, P240, P212, P208, P114, P217, P204, P213 and P222

Investigator: Johanna, Andi

Aim of the experiment: Retransformation of E. coli XL1 blue with P170, P111, P314, P160, P275, P244, P240, P212, P208, P114, P217, P204, P213 and P222.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 2 µl of ddH2O was added to empty tubes
  • 2 µl of DNA was added to 100 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The transformated cells were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on chlorampenicol (P170, P111, P160, P275, P244, P240, P212, P208, P217, P204, P213, P222) and ampicillin plates (P314, P114).

Sequencing of P320 (ReTrafo of P238,SERK-TMD_8AAlinker_GFPmut1)

Investigators: Rosario

Aim of the experiment: Sequencing of P320 (ReTrafo of P238,SERK-TMD_8AAlinker_GFPmut1).

Procedure: Peparation of DNA for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng/µl) and 2 µl sequencing primer 10 µM).


The different genes received the following barcodes:

Label Name Barcode1 Barcode2
P308 SERK-TMD_8AAlinker_GFPmut1 FR02305808 (O3) FR02305807 (O4)

Preparation of media for competent cells

Investigator : Johanna, Louise

Aim of the experiment:Preparation of media for competent cells

Procedure:

  • Picking of one colony of overnight stem culture of non competent XL1-Blue E.Coli in 50 ml LB-Medium without antibiotics (under the sterile bench)
  • this was incubate overnight at 37 °C
  • Batch for 1l of a 0.1 M MgCl2-solution
amount reagent
20.33 g MgCl2 (M=203.3 g/mol)
1 l ELGA H2O
  • Batch for 500ml of a 50 mM CaCl2-solution
amount reagent
3.67 g CaCl2 (M=147.02 g/mol)
500 ml ELGA H2O
  • Batch for 50ml of a 50 mM CaCl2-solution, 15% v/v Glycerin
amount reagent
0.368 g CaCl2 (M=147.02 g/mol)
0.458 g Glycerin (density=1.26)
50 ml ELGA H2O
  • Autoclave each bottle of solution and leave each in a 4°C room.

QuikChanges QC pActin and QC1 AlcR

Investigator : Andi

Aim of the experiment: QuikChanges QC pActin (P114) and QC1 AlcR (P294)

Procedure: Quickchange - PCR

Reaction batch 1 for P114 (pActin) with Pfu Ultra II

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid template (P114 undiluted) 4.5 ng in 25 µl batch
1 µl 1:10 dilution of O76 (=10 µM) 0.5 µl Primer + 4.5 µl ddH2O
1 µl 1:10 dilution of O77 (=10 µM) 0.5 µl Primer + 4.5 µl ddH2O
18 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)

PCR cycling parameters - P114 (pActin)

Segment Cycles Temperature Time
1 1 95 °C 30 s
2 20 95 °C 30 s
3 52 °C 1 min
4 68 °C 6.5 min
5 4 °C hold

Reaction batch 2 for P114 (pActin) with Q5 Polymerase Mastermix

volume reagent Additional information
12.5 µl 2x QS Polymerase Mastermix
1 µl Plasmid template (P114 - 1:10 dilution) 0.45 ng in 25 µl batch
1.25 µl 1:10 dilution of O76 (=10 µM) 0.5 µl Primer + 4.5 µl ddH2O
1.25 µl 1:10 dilution of O77 (=10 µM) 0.5 µl Primer + 4.5 µl ddH2O
9 µl ddH2O

PCR cycling parameters - P114 (pActin) with Q5 Polymerase

Segment Cycles Temperature Time
1 1 95 °C 30 s
2 20 95 °C 30 s
3 55 °C 1 min
4 72 °C 3.25 min
5 4 °C hold

Reaction batch for P294 (AlcR) with Pfu Ultra II

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
0.5 µl Plasmid template (P294 dilutsion 1:100) 2.79 ng in 25 µl batch
1 µl 1:10 dilution of O76 (=10 µM) 0.5 µl Primer + 4.5 µl ddH2O
1 µl 1:10 dilution of O77 (=10 µM) 0.5 µl Primer + 4.5 µl ddH2O
18.5 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)

PCR cycling parameters - P294 (AlcR)

Segment Cycles Temperature Time
1 1 95 °C 30 s
2 20 95 °C 30 s
3 52 °C 1 min
4 68 °C 6.5 min
5 4 °C hold
  • After the PCR, the Quickchange products of P114 (pActin) with Q5 Mastermix and P294 (AlcR)were mixed with 1 µl of DpnI (10 U/µl) and incubated at 37 °C for 1 h.
  • After the PCR, the Quickchange products of P114 (pActin) with PfU II Polymerase was stored in the Cycler at 4 °C over night


Transformation of E. coli XL1-blue with DpnI digested QuikChange product P114 (pActin) and P294 (AlcR)

Investigator: Master of Quickchange, Gel-Penetrator

Aim of the experiment: Transformation of E. coli XL1-blue with DpnI digested QuikChange product P294 (AlcR) QCI and P114 (pActin).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of QuikChange products/2 µl of plasmid DNA was added to 200 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol/ampicillin plate.


PCR of P317 (Laccase) with Q5 Mastermix and Herculase Polymerase

Investigator: Andi, Johanna

Aim of the experiment: PCR of P317 (Laccase) with Q5 Mastermix and Herculase Polymerase

Procedure:

Operational sequence:

  • Reaction batch with Herculase Polymerase
volume reagent
10 µl 5x Herculase Fusion Polymerase reaction buffer
2.5 µl 10 mM dNTPs
1.25 µl 10 µM Forward Primer O24 (Bpul_for)
1.25 µl 10 µM Reverse Primer O25 (Bpul_rev)
1 µL Herculase II Fusion Polymerase
1.5 µl Plasmid DNA (P317)- 1:500 dilution
0.5 µl DMSO
32 µL ddH2O Water
=50 µL TOTAL
  • Reaction batch with Q5 Mastermix
volume reagent
25 µl 2x Q5 Master Mix
2.5 µl 10 µM Forward Primer O24 (Bpul_for)
2.5 µl 10 µM Reverse Primer O25 (Bpul_rev)
1 µl Plasmid DNA (P317)- 1:500 dilution
19 µL ddH2O Water
=50 µL TOTAL
  • The content has been mixed with a pipette
  • The PCR program was performed after following scheme:
Initial denaturation 94 °C 30 s
30 cycles 94 °C 30 s
51 °C 60 s
68 °C 90 s
Final extension 68 °C 5 min
Hold 4 °C infinite
  • After the PCR has finished, the product was hold at 4 °C in the Cycler

Analytical gelelectrophoresis of the DpnI digested products of the performed QuickChange of SDMI of P294 (AlcR)and P114 (pActin)

Investigator: Andi, Johanna

Aim of the experiment: Analytical gelelectrophoresis of the DpnI digested products of the performed QuikChange of SDMI of P294 (AlcR)and P114 (pActin.)

Procedure:

  • 9 µl of each of the DpnI digested QuikChange products were mixed with 1 µl of DNA loading buffer (10x).
  • Analytical gelelectrophoresis was performed at 90 V for 50 min at an 1% agarose gel.

TUM13 20130620 QC AlcR QC pActin.png

Lane: 100 bp DNA ladder DpnI digestion of P294QCI DpnI digestion of P114QCI 100 bp DNA ladder
Result: expected result only Primers

Analytical digestion and gelelectrophoresis of P315 and P316 (QC I P162 NPT-casette), P162 (npt-casette in pSB1C3), P317 and P318 (QC V P217 Laccase), P217 (Laccase), P322 (F77+F105 NucA+NLS), P248 (Thermonuc in pSB1C3), P320 (SERK-TMD_8AAlinker_GFPmut1), P326+P327 (Strep-tag-TEV-site-linker+SERK-TMD_8AAlinker_GFPmut1), P328+P329 (SERK-SigP+EreB), P157 (EreB), P330+P331 (SERK-SigP+NanoLuc), P208 (NanoLuc), P332+P333 (SERK-SigP+SpyCatcher), P213 (SpyCatcher), P334+335 (SERK-SigP+SpyTag), P170 (SpyTag), P336+P337 (SERK-SigP+xylE), P197 (xylE), P338+P339 (SERK-SigP+SERK-TMD), P212(SERK-TMD)

Investigator: Rosario, Katrin

Aim of the experiment: Analytical digestion and gelelectrophoresis of P315 and P316 (QC I P162 NPT-casette), P162 (npt-casette in pSB1C3), P317 and P318 (QC V P217 Laccase), P217 (Laccase), P322 (F77+F105 NucA+NLS), P248 (Thermonuc in pSB1C3), P320 (SERK-TMD_8AAlinker_GFPmut1), P326+P327 (Strep-tag-TEV-site-linker+SERK-TMD_8AAlinker_GFPmut1), P328+P329 (SERK-SigP+EreB), P157 (EreB), P330+P331 (SERK-SigP+NanoLuc), P208 (NanoLuc), P332+P333 (SERK-SigP+SpyCatcher), P213 (SpyCatcher), P334+335 (SERK-SigP+SpyTag), P170 (SpyTag), P336+P337 (SERK-SigP+xylE), P197 (xylE), P338+P339 (SERK-SigP+SERK-TMD), P212(SERK-TMD)

Procedure:

  • Batch
volume reagent
2 µl CutSmart Buffer (10x)
0.25 µl EcoRI
0.25 µl PstI
15 µl ddH2O
2.5 µl Plasmid DNA
=20 µl TOTAL
  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min

TUM13 20130620 anal Verd P315 P316 P162 P317 P318 P217 P322-P325 P248 PstI.EcoRI.png

1 kb DNA Ladder P315 P316 P162 (Control) P317 P318 P217 (Control) P322 P323 P324 P325 P248 (Control)
as expected as expected as expected as expected as expected as expected corrupt corrupt corrupt corrupt as expected

TUM13 20130620 anal Verd P334 P335 P170 P336 P337 P197 P338 P339 P212 P320 PstI.EcoRI.png


1 kb DNA Ladder P334 P335 P170 (Control) P336 P337 P197 (Control) P338 P339 P212 (Control) P320
as expected as expected as expected as expected as expected as expected as expected as expected as expected is repeated on other gel

TUM13 20130620 anal Verd P320 326 327 PstI.EcoRI.png

1 kb DNA Ladder P320 (Control) P326 P327
as expected as expected as expected

TUM13 20130620 anal Verd P328 P329 P157 P330 P331 P208 P332 P333 P213 P326 P327 PstI.EcoRI.png

1 kb DNA Ladder P328 P329 P157 (Control) P330 P331 P208 (Control) P332 P333 P213 (Control) P326 P327
as expected as expected as expected as expected as expected corrupt as expected as expected corrupt is repeated on other gel is repeated on other gel

Friday, June 21st

Analytical gelelectrophoresis of the PCR product of P317 (Laccse) with PfU II Polymerase, Herculase Polymerase and the DpnI digested products of the performed QuickChange of P114 (pActin)

Investigator: Master of QuikChange

Aim of the experiment: Analytical gelelectrophoresis of the PCR product of P317 (Laccse) with PfU II Polymerase, Herculase Polymerase and the DpnI digested products of the performed QuickChange of P114 (pActin)

Procedure:

  • 9 µl of each of the PCR products were mixed with 1 µl of DNA loading buffer (10x).
  • 9 µl of each of the DpnI digested QuikChange product was mixed with 1 µl of DNA loading buffer (10x).
  • Analytical gelelectrophoresis was performed at 90 V for 50 min at an 1% agarose gel.

TUM13 20130621 F111 F112 QC P114.png

Lane: 1000 bp DNA ladder PCR product of P317 with Q5 Master Mix PCR product of P317 with Herculase Polymerase DpnI digestion of P114
Result: expected result expected result expected result

Preparation of competent cells according to the CaCl 2-method (Cohen et al.,1972)

Investigator: Johanna, Louise


Aim of the experiment: Preparation of competent cells according to the CaCl 2-method (Cohen et al.,1972)

Procedure:

  • Add Tetracyclin (ratio: 1:1000) to the 2YT-medium
  • Measure the OD550 of it until OD550=0.5
  • Put the culture in Falcon tubes (leaving on ice)
  • Centrifuge the falcon tubes by 5000 rpm, 4°C, 10 min
  • Resuspend the falcon tubes each with 40 ml 0,1 M MgCl2 solution after removing the supernatants
  • Centrifuge the falcon tubes by 5000 rpm, 4°C, 10 min
  • Resuspend the falcon tubes each with 20 ml 50mM CaCl2 solution after removing the supernatants
  • Incubate the falcon tubes on ice for 30 min
  • Centrifuge the falcon tubes by 5000 rpm, 4°C, 10 min
  • Resuspend the falcon tubes each with 2 ml 50mM CaCl2, 15 % v/v Glycerin solution after removing the supernatants. From this step everything needs to be carry out in a 4°C room.
  • Mix the solutions of each falcon tube and transfer the content in eppis (each with 100 and accordingly 150 µl)
  • Store the boxes of eppis at -80 °C

Preparative Digestion of P308 (PhyB-Linker-C-TEV), P299 (IRES), P111 (TEV), P326 (TEV-Linker-TM-GFP), P328 (SERKSigP_EreB), P330 (SERK-SigP_NLuc), P332 (SERK-SigP_SpyCatcher), P334 (SERK-SigP_SpyTag), P336 (SERK-SigP_XylE), P338 (SERK-SigP_SERK-TMD), P8 (pSB1C3) and PCR product of Laccase (F112)

Investigator: Katrin

Aim of the experiment: Peparative Digestion of P308 (PhyB-Linker-C-TEV), P299 (IRES), P111 (TEV), P326 (TEV-Linker-TM-GFP), P328 (SERKSigP_EreB), P330 (SERK-SigP_NLuc), P332 (SERK-SigP_SpyCatcher), P334 (SERK-SigP_SpyTag), P336 (SERK-SigP_XylE), P338 (SERK-SigP_SERK-TMD) and P8 (PhyB)

Procedure:

Batch for preparative digestion of effectors (P328, P330, P332, P334, P336, P338) with AgeI and PstI) (7x Mastermix)

volume reagent
28 µl Buffer 4 (10x)
7 µl AgeI (20 U/µl)
7 µl PstI (20 U/µl)
98 µl ddH2O
=140 µl TOTAL
  • 20 µl Mastermix + 20 µl Plasmid each

Batches for preparative digestions:

volume reagent
20 µl Plasmid
4 µl Buffer 4 (10x)
1 µl Enzyme 1
1 µl Enzyme 2
14 µl ddH2O
=40 µl TOTAL
Plasmid Name Enzyme 1 Enzyme 2
P308 PhyB-Linker-C-TEV SpeI PstI
P299 IRES XbaI PstI
P111 TEV SpeI PstI
P326 TEV-Linker-TM-GFP NgoMIV PstI
P8 pSB1C3 XbaI AgeI
F112 Laccase XbaI AgeI
  • Incubation for 2.5 h at 37 °C.
  • 4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and they were loaded on a 1% agarose gel
  • Preparative gelelectrophoresis was performed at 90 V for 60 min.

TUM13 20130621 prep Verd. P308 SpeI.PstI P299 XbaI.Pst P111 SpeI.PstI.png

Name: P308 dig. SpeI + PstI 1kb DNA ladder P299 dig. XbaI + PstI P111 dig. SpeI + PstI
cut out: Backbone Insert (lower band) Backbone


TUM13 20130621 prep Verd. P326 NgoMIV .PstI P328 AgeI.Pst P330 AgeI.PstI.png

Name: P326 dig. NgoMIV + PstI 1kb DNA ladder P328 dig. AgeI + Pst P330 dig. AgeI + PstI
cut out: Insert (lower band) Backbone Backbone

TUM13 20130621 prep Verd. P332 AgeI.Pst P334 AgeI.Pst P336 AgeI.PstI.png

Name: P332 dig. AgeI + PstI 1kb DNA ladder P334 dig. AgeI + PstI P336 dig. AgeI + PstI
cut out: Backbone Backbone Backbone

TUM13 20130621 prep Verd. P338 AgeI.Pst P8 AgeI.XbaI.png

Name: P338 dig. AgeI + PstI 1kb DNA ladder P8 dig. AgeI + XbaI
cut out: Backbone Backbone (lower band)
  • Extracted via QIAGEN gel extraction kit, digested PCR product purified via PCR Purification Kit


Ligation of F116+F117 (TEV-Linker-TM-GFP+SERKSigP_EreB), F116+F118 (TEV-Linker-TM-GFP+SERK-SigP_NLuc), F116+F119 (TEV-Linker-TM-GFP+SERK-SigP_SpyCatcher), F116+F120 (TEV-Linker-TM-GFP+SERK-SigP_SpyTag), F116+F121 (TEV-Linker-TM-GFP+SERK-SigP_XylE), F123+F124 (Laccase+pSB1C3)

Investigator: Katrin

Aim of the experiment: Ligation of F116+F117 (TEV-Linker-TM-GFP+SERKSigP_EreB), F116+F118 (TEV-Linker-TM-GFP+SERK-SigP_NLuc), F116+F119 (TEV-Linker-TM-GFP+SERK-SigP_SpyCatcher), F116+F120 (TEV-Linker-TM-GFP+SERK-SigP_SpyTag), F116+F121 (TEV-Linker-TM-GFP+SERK-SigP_XylE), F123+F124 (Laccase+pSB1C3)

Procedure:

  • Ligation batch for F116+F117 (TEV-Linker-TM-GFP+SERKSigP_EreB)
volume reagent
3.52 µl F117 (28.4 ng/µl, 3413 bp)
4.81 µl F116 (18.2 ng/µl, 996 bp)
8.67 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F116+F118 (TEV-Linker-TM-GFP+SERK-SigP_NLuc)
volume reagent
1.89 µl F118 (52.8 ng/µl, 2663 bp)
6.16 µl F116 (18.2 ng/µl, 996 bp)
8.95nbsp;µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL


  • Ligation batch for F116+F119 (TEV-Linker-TM-GFP+SERK-SigP_SpyCatcher)
volume reagent
2.57 µl F119 (38.0 ng/µl, 2492 bp)
6.59 µl F116 (18.2 ng/µl, 996 bp)
7.84nbsp;µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F116+F120 (TEV-Linker-TM-GFP+SERK-SigP_SpyTag)
volume reagent
0.8 µl F120 (62.5 ng/µl, 2192 bp)
3.75 µl F116 (18.2 ng/µl, 996 bp)
3.96nbsp;µl ddH2O
1 µl T4 ligase buffer (10x)
0.5 µl T4 ligase (1 U/µl)
=10 µl TOTAL


  • Ligation batch for F116+F121 (TEV-Linker-TM-GFP+SERK-SigP_XylE)
volume reagent
0.73 µl F121 (68.3 ng/µl, 3071 bp)
2.56 µl F116 (18.2 ng/µl, 996 bp)
5.95nbsp;µl ddH2O
1 µl T4 ligase buffer (10x)
0.5 µl T4 ligase (1 U/µl)
=10 µl TOTAL


  • Ligation batch for F123+F124 (Laccase+pSB1C3)
volume reagent
2.03 µl F123 (49.3 ng/µl, 2086 bp)
3.80 µl F124 (58.9 ng/µl, 1555 bp)
11.17nbsp;µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Negative control was prepared by replacing the volume of the insert by ddH2O.
  • The ligation was performed for 1 hour at room temperature.


Transformation of E. coli XL1-blue with ligation products F116+F117 (TEV-Linker-TM-GFP+SERKSigP_EreB), F116+F118 (TEV-Linker-TM-GFP+SERK-SigP_NLuc), F116+F119 (TEV-Linker-TM-GFP+SERK-SigP_SpyCatcher), F116+F120 (TEV-Linker-TM-GFP+SERK-SigP_SpyTag), F116+F121 (TEV-Linker-TM-GFP+SERK-SigP_XylE), F123+F124 (Laccase+pSB1C3) and negative controls

Investigator: Jeff, Katrin

Aim of the experiment: Transformation of E. coli XL1-blue with ligation products F116+F117 (TEV-Linker-TM-GFP+SERKSigP_EreB), F116+F118 (TEV-Linker-TM-GFP+SERK-SigP_NLuc), F116+F119 (TEV-Linker-TM-GFP+SERK-SigP_SpyCatcher), F116+F120 (TEV-Linker-TM-GFP+SERK-SigP_SpyTag), F116+F121 (TEV-Linker-TM-GFP+SERK-SigP_XylE), F123+F124 (Laccase+pSB1C3), QCI(Insertion) of pActin and negative controls


Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of QuikChange products/2 µl of plasmid DNA was added to 150 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol/ampicillin plate.

Sequencing of P303 (SERK-SigP in PsB1C3, fw), P310 (N-TEV_36AALinker in pSB1C3, rev), P248 (Thermonuc in pSB1C3, fw), P312 (N-TEV_36AALinker in pSB1C3, fw), P316 (npt-casette after QCI, fw,rev), P318 (Laccase after QCV, still RFC10, fw, rev), P320 (SERK-TMD_8AAlinker_GFPmut1, fw, rev), P326 (Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1, fw, rev), P328 (SERK-SigP_EreB, fw, rev), P330 (SERK-SigP_NanoLuc, fw), P332 (SERK-SigP_SpyCatcher, fw), P334 (SERK-SigP_SpyTag, fw), P336 (SERK-SigP_XylE, fw, rev), P338 (SERK-SigP_SERK-TMD, fw)

Investigators: Louise, Katrin

Aim of the experiment: Sequencing of P303 (SERK-SigP in PsB1C3, fw), P310 (N-TEV_36AALinker in pSB1C3, rev), P248 (Thermonuc in pSB1C3, fw), P312 (N-TEV_36AALinker in pSB1C3, fw), P316 (npt-casette after QCI, fw,rev), P318 (Laccase after QCV, still RFC10, fw, rev), P320 (SERK-TMD_8AAlinker_GFPmut1, fw, rev), P326 (Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1, fw, rev), P328 (SERK-SigP_EreB, fw, rev), P330 (SERK-SigP_NanoLuc, fw), P332 (SERK-SigP_SpyCatcher, fw), P334 (SERK-SigP_SpyTag, fw), P336 (SERK-SigP_XylE, fw, rev), P338 (SERK-SigP_SERK-TMD, fw).

Procedure: Peparation of DNA for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng/µl) and 2 µl sequencing primer 10 µM).


The different genes received the following barcodes:

Label Name Barcode1 Barcode2
P303 SERK-SigP FR02305805 (O3)
P310 N-TEV_36AAlinker FR02305806 (O4)
P248 Thermonuc. FR02305804 (O3)
P312 NTEV_36aaLinker FR02305803 (O3)
P316 npt-casette QCI FR02305802 (O3) FR02305801 (O4)
P318 Laccase QCV FR02305800 (O3) FR02305799 (O4)
P320 SERK-TM_8aaLinker_GFPmut1 FR02305798 (O3) FR02305797 (O4)
P326 Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 FR02305796 (O3) FR02305795 (O4)
P328 SERK-SigP_EreB FR02305794 (O3) FR02305793 (O4)
P330 SERK-SigP_Nanoluc FR02305792 (O3)
P332 SERK-SigP_Spycatcher FR02305791 (O3)
P334 SERK-SigP_Spy-tag FR02305790 (O3)
P336 SERK-SigP_XylE FR02305789 (O3) FR02305788 (O4)
P338 SERK-SigP_SERK-TM FR02305787 (O3)

PCR of P61 to get the IRES of poliovirus 1 Mahoney strain

Investigator: Jeff

Aim of the experiment: PCR of P61 to get the IRES of poliovirus 1 Mahoney strain.

Procedure:

Operational sequence:

  • Reaction batch with Q5 Mastermix:
volume reagent
25 µl 2x Q5 Master Mix
2.5 µl 10 µM Forward Primer O68 (IRES_polio_fw)
2.5 µl 10 µM Reverse Primer O69 (IRES_polio_rv)
1 µl Plasmid DNA (P61)- 1:1000 dilution (desired c between 1 pg and 1 ng)
19 µL ddH2O Water
=50 µL TOTAL
  • The content has been mixed with a pipette
  • The PCR program was performed after following scheme:
Initial denaturation 98 °C 30 s
30 cycles 98 °C 10 s
63 °C 30 s
72 °C 20 s
Final extension 72 °C 2 min
Hold 4 °C infinite
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

Picking of Re-Transformations P111 (TEV in pSB1C3, Re-Trafo), P160 (Sv40 NLS in pSB1C3), P114 (pActin), P314 (FluA), Glutathione S-transferase (GST, BBa_K620000), QCI AlcR

Investigator: Katrin

Aim of the experiment: Picking of Re-Transformations P111 (TEV in pSB1C3, Re-Trafo), P160 (Sv40 NLS in pSB1C3), P114 (pActin), P314 (FluA), Glutathione S-transferase (GST, BBa_K620000), QCI AlcR.

Procedure:

  • Retransformations 1 clone each
  • Resuspendsion in 4 ml LB-medium with 4 µl chloramphenicol (P111, P160) or ampicillin (P114, P314)

Saturday, June 22nd

QuikChange of npt-casette (P316, QCII) and of AlcR (P342, QCII)

Investigator: Jeff

Aim of the experiment: QuikChange of npt-casette (P316, QCII) and of AlcR (P342, QCII).

Procedure:

Reaction batch for P316 (npt-casette after QCI)

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid template (P316 - 1:100 dilution) need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl 1:10 dilution of O72 (=10 µM)
1 µl 1:10 dilution of O73 (=10 µM)
19 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)

Reaction batch for P342 (AlcR after QCI):

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid template (P342 - 1:100 dilution) need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl 1:10 dilution of O82 (10 µM)
1 µl 1:10 dilution of O83 (10 µM)
19 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)

PCR cycling parameters - P316 (npt-casette after QCI) and P342 (AlcR after QCI):

Segment Cycles Temperature Time
1 1 95 °C 30 s
2 20 95 °C 30 s
52 °C 1 min
68 °C 5 min
3 4 °C hold

Miniprep of overnight cultures of transformated E. coli XL1 blue with P111(Retrafo), QCI AlcR, P114(Retrafo), P314 (Retrafo), P160 (Retrafo), Glutathione S-transferase (GST, BBa_K620000)

Investigator: Katrin

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with P111(Retrafo), QCI AlcR, P114(Retrafo), P314 (Retrafo), P160 (Retrafo), Glutathione S-transferase (GST, BBa_K620000).

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
  • The resulting DNA were eluted in tubes labeled as follows:

Analytical digestion and gelelectrophoresis of QCII AlcR (P341, P342) and control P294 with EcoRI and PstI, gelelectrophoresis of PCR of IRES

Investigator: Katrin

Aim of the experiment: Analytical digestion and gelelectrophoresis of QCII AlcR (P341, P342) and control P294 with EcoRI and PstI

Procedure:

  • Batch
volume reagent
2 µl CutSmart Buffer (10x)
0.25 µl EcoRI
0.25 µl PstI
15 µl ddH2O
2.5 µl Plasmid DNA (P341, P342; P249)
=20 µl TOTAL
  • Incubation for 60 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min

TUM13 20130622 anal Verd. F124(IRES) P294 P341 P342 EcoRI.PstI.png

Lane: 1 kb DNA Ladder PCR of IRES Digestion of P249 with EcoRI and PstI (control) Digestion of P341 with EcoRI and PstI Digestion of P342 with EcoRI and PstI
Result: as expected as expected as expected as expected


Preparative Digestion of P310 (N-Tev-linker), P249 (Thermonuclease), P160 (SV40 NLS), PCR of IRES

Investigator: Katrin

Aim of the experiment: Preparative Digestion of P310 (N-Tev-linker), P249 (Thermonuclease), P160 (SV40 NLS), PCR of IRES

Procedure:

Preparative digestion of P310 (N-Tev-linker)

volume reagent
20 µl P310
4 µl CutSmart Buffer (10x)
1 µl AgeI (20 U/µl)
1 µl SpeI (20 U/µl)
14 µl ddH2O
=40 µl TOTAL

Preparative digestion of P160 (SV40 NLS)

volume reagent
20 µl P160
4 µl CutSmart Buffer (10x)
1 µl AgeI (20 U/µl)
1 µl PstI (20 U/µl)
14 µl ddH2O
=40 µl TOTAL


Preparative digestion of P249 (Thermonuclease)

volume reagent
20 µl P249
4 µl CutSmart Buffer (10x)
1 µl NgoMIV (20 U/µl)
1 µl PstI (20 U/µl)
14 µl ddH2O
=40 µl TOTAL


Preparative digestion of PCR of IRES)

volume reagent
25 µl PCR product
5 µl CutSmart Buffer (10x)
1 µl EcoRI (20 U/µl)
1 µl SpeI (20 U/µl)
18 µl ddH2O
=50 µl TOTAL
  • Incubation for 2.5 h at 37 °C.
  • 4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and they were loaded on a 1% agarose gel
  • Preparative gelelectrophoresis was performed at 90 V for 60 min.

TUM13 20130622 prep Verd. P249 NgoMIV.PstI P160 AgeI.PstI P310 AgeI.SpeI.png

Name: P249 dig. NgoMIV + PstI 1kb DNA ladder P160 dig. AgeI + PstI P111 dig. SpeI + AgeI
cut out: Insert(lower band) Backbone Backbone
  • Extracted via QIAGEN gel extraction kit, digested PCR product purified via PCR Purification Kit

Ligation of F126+F127 (NLS in pSB1C3+Thermonuclease), F125+F96 (N-TEV-linker in pSB1C3+PIF3), F125+F98 (N-TEV-linker in pSB1C3+PIF6), F124+F41 (IRES+pSB1C3)

Investigator: Katrin

Aim of the experiment: Ligation of F126+F127 (NLS in pSB1C3+Thermonuclease), F125+F96 (N-TEV-linker in pSB1C3+PIF3), F125+F98 (N-TEV-linker in pSB1C3+PIF6), F124+F41 (IREs+pSB1C3)

Procedure:

  • Ligation batch for F126+F127 (NLS in pSB1C3+Thermonuclease
volume reagent
7.2 µl F126 (13.9 ng/µl, 2143 bp)
4.1 µl F127 (15.2 ng/µl, 447 bp)
5.7 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F125+F96 (N-TEV-linker in pSB1C3+PIF3)
volume reagent
6.9 µl F125 (14.5 ng/µl, 2554 bp)
2.3 µl F96 (15.1 ng/µl, 297 bp)
7.8nbsp;µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F124+F41 (IREs+pSB1C3)
volume reagent
1.3 µl F124 (70.8 ng/µl, 628 bp)
1.2 µl F41 (81.1 ng/µl, 2086 bp)
14.5nbsp;µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Negative control was prepared by replacing the volume of the insert by ddH2O.
  • The ligation was performed for 1 hour at room temperature.


Transformation of E. coli XL1-blue with ligation products F126+F127 (NLS in pSB1C3+Thermonuclease), F125+F96 (N-TEV-linker in pSB1C3+PIF3), F125+F98 (N-TEV-linker in pSB1C3+PIF6), F124+F41 (IRES+pSB1C3) and negative controls

Investigator: Volker

Aim of the experiment: Transformation of E. coli XL1-blue with ligation products F126+F127 (NLS in pSB1C3+Thermonuclease), F125+F96 (N-TEV-linker in pSB1C3+PIF3), F125+F98 (N-TEV-linker in pSB1C3+PIF6), F124+F41 (IRES+pSB1C3) and negative controls


Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of QuikChange products/2 µl of plasmid DNA was added to 150 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Picking of F116+F121, F116+F117, F123+F124, F116+F119, F116+F120, F116+F118, QCI (insertion of P114 (pActin)

Investigator: Katrin Aim of the experiment: Picking of F116+F121, F116+F117, F123+F124, F116+F119, F116+F120, F116+F118, QCI (insertion of P114 (pActin)

Procedure:

  • 2 clones were picked from each plate
  • Resuspendsion in 4 ml LB-medium with 4 µl chloramphenicol or ampicillin (QCI (insertion of P114 (pActin))

Sunday, June 23rd

QuikChange II of pActin (P359, P360)

Investigator: Katrin

Aim of the experiment: QuikChangeII of pActin (P359, P360) Procedure:

Reaction batch

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid template (P359, P360) need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl 1:10 dilution of O76 (=10 µM)
1 µl 1:10 dilution of O77 (=10 µM)
19 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)

PCR cycling parameters

Segment Cycles Temperature Time
1 1 95 °C 30 s
2 20 95 °C 30 s
52 °C 1 min
68 °C 6 min
3 4 °C hold

Miniprep of overnight cultures of transformated E. coli XL1 blue F123+F124, F116+F117, F116+F119, F116+F118, F116+F121, F116+F120, QCI pActin (P114)

Investigator: Katrin

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue F123+F124, F116+F117, F116+F119, F116+F118, F116+F121, F116+F120, QCI pActin (P114)

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)


Analytical digestion and gelelectrophoresis of P347-P358

Investigator: Katrin

Aim of the experiment: Analytical digestion and gelelectrophoresis of P347-P358

Procedure:

volume reagent
2 µl CutSmart Buffer (10x)
0.25 µl EcoRI
0.25 µl PstI
15 µl ddH2O
2.5 µl Plasmid DNA (P347-P358)
=20 µl TOTAL
  • Incubation for 60 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min

TUM13 20130623 anal Verd. P347-P354 EcoRI.PstI.png

Lane: 1 kb DNA Ladder Digestion of P347 with EcoRI and PstI Digestion of P348 with EcoRI and PstI Digestion of P349 with EcoRI and PstI Digestion of P350 with EcoRI and PstI Digestion of P351 with EcoRI and PstI Digestion of P352 with EcoRI and PstI Digestion of P353 with EcoRI and PstI Digestion of P354 with EcoRI and PstI
Result:


TUM13 20130623 anal Verd. P355-P358 EcoRI.PstI.png

Lane: 1 kb DNA Ladder Digestion of P355 with EcoRI and PstI Digestion of P356 with EcoRI and PstI Digestion of P357 with EcoRI and PstI Digestion of P358 with EcoRI and PstI
Result:

Dpn1 Digestion and transformation of E. coli XL1-blue with QCII of AlcR and QCII of npt-cassette

Investigator: Katrin

Aim of the experiment: Dpn1 Digestion and transformation of E. coli XL1-blue with QCII of AlcR and QCII of npt-cassette

Procedure:

  • 1 µl of Dpn1 was added to the reaction, the batch was incubated for one hour at 37°C
  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of QuikChange products was added to 150 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Picking of F125+F98, F125+F96, F128+F41

Investigator: Katrin Aim of the experiment:Picking of F125+F98, F125+F96, F128+F41

Procedure:

  • 2 clones were picked from each plate
  • Resuspendsion in 4 ml LB-medium with 4 µl chloramphenicol
  • F126+F127 was put back into the incubator because there were no colonies visible

Sequencing of P35, P143, P359, P360, P249

Investigators: Katrin

Aim of the experiment: Sequencing of P35, P143, P359, P360, P249

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:


Label Barcode1 Barcode2
P35 FR02305786 (O4)
P143 FR02305785 (O92) FR02305784 (O93)
P359 FR02305783 (O62) FR02305782 (O63)
P360 FR02305781 (O62) FR02305780 (O63)
P249 FR02305779 (O3) FR02305778 (O4)

Week 10

Monday, June 24th

Miniprep of ligations F125+F98 (N-TEV-linker in pSB1Cs + PIF6), F125+F96 (N-TEV-linker in pSB1Cs + PIF3) and F128+F41 (PCR of IRES + pSB1C3 backbone) + analytical gel

Investigator: Johanna

Aim of the experiment: Preparation of ligation products F125+F98, F125+F96 and F128+F41

Procedure:

  • Miniprep according to QIAGEN QIAprep Kit
label content conc. in ng/µl
P361 N-TEV-linker (in pSB1C3) + PIF6 163.2
P362 N-TEV-linker in (in pSB1C3) + PIF6 183.5
P363 N-TEV-linker in (in pSB1C3) + PIF3 159.6
P364 N-TEV-linker in (in pSB1C3) + PIF3 200.1
P365 PCR of IRES + (in pSB1C3) backbone 136.0
P366 PCR of IRES + (in pSB1C3) backbone 153.7
  • Analytical digestion with EcoRI and PstI
reagent volume
CutSmart buffer 2 µl
EcoRI 0.25 µl
PstI 0.25 µl
ddH2O 15 µl
plasmid DNA 2.5 µl
Total 20 µl
  • 60 min incubation, 37 °C
  • Gel elektrophoresis on 1% agarose gel

TUM13 20130624 anal Gel P361-P366 EcoRI.PstI.png

1 kbp DNA ladder P361 P362 P363 P364 P365 P366
as expected as expected as expected as expected as expected as expected

Dpn1 digestion and transformation of QCII of pActin (insertion) + analytical Gel

Investigator: Jeff

Aim of the experiment: Dpn1 digestion and transformation of QCII of pActin (insertion)

Procedure:

  • 1 µl of Dpn1 (10 U/µl) was added to the reaction, the batch was incubated for one hour at 37°C
  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 8 µl of QuikChange products was added to 150 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new ampicillin plate.
  • 0.5 µl of loading buffer were added to 4.5 µl of Dpn1 digested plasmid
  • Electrophoresis on 1% agarose gel

TUM13 20130624 anal Gel P359QCII P360QCII.png

1 kb dna ladder P359 P360
successful successful

Sequencing of P266 (IgKappa-SigP_Nanoluc), P268 (IgKappa-SigP_XylE), P270 (IgKappa-SigP_SpyCatcher), P272 (IgKappa-SigP_SpyTag), P361 (N-TEV-linker+PIF6), P364 (N-TEV-36AAlinker-PIF3) and P366 (IRES)

Investigator: Florian

Aim of the experiment: Sequencing of P266 (IgKappa-SigP_Nanoluc), P268 (IgKappa-SigP_XylE), P270 (IgKappa-SigP_SpyCatcher), P272 (IgKappa-SigP_SpyTag), P361 (N-TEV-linker+PIF6), P364 (N-TEV-36AAlinker-PIF3) and P366 (IRES)

Procedure: DNA samples were prepared according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The samples received the following barcodes:

Label Barcode1 Barcode2
P266 FR02305766 fw FR02305767 rv
P268 FR02305768 fw FR02305769 rv
P270 FR02305770 fw
P272 FR02305771 fw
P361 FR02305772 fw FR02305773 rv
P364 FR02305774 fw FR02305775 rv
P366 FR02305776 fw FR02305777 rv

Retransformation of P270 (IgKappa-SigP_SpyCatcher), P272 (IgKappa-SigP_SpyTag), P308 (PhyB_36AALinker_C-TEV) and P366 (IRES)

Investigator: Johanna

Aim of the experiment: Retransformation of P270 (IgKappa-SigP_SpyCatcher in pSB1C3), P272 (IgKappa-SigP_SpyTag in pSB1C3), P308 (PhyB_36AALinker_C-TEV in pSB1C3) and P366 (IRES in pSB1C3)

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 1.5 µl of plasmid DNA was added to 150 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of cell suspension were plated on chlorampenicol agar
  • The rest of the cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium; the concentrated cell suspension was plated on another chlorampenicol agar plate.

Ligation of F92 + F130 (IgKappa-SigP + Laccase), F109 + F130 (SERK-SigP + Laccase), F113 + F131 (PhyB_36AA-Linker_C-TEV + IRES), F115 + F131 (TEV + IRES)

Investigator: Florian, Jeff

Aim of the experiment: Ligation of F92 + F130 (IgKappa-SigP + Laccase), F109 + F130 (SERK-SigP + Laccase), F113 + F131 (PhyB_36AA-Linker_C-TEV + IRES), F115 + F131 (TEV + IRES).

Procedure:

  • Ligation batch for F92 + F130 (IgKappa-SigP + Laccase)
volume reagent
0.86 µl F92 (116.7 ng/µl, 2144 bp)
7.55 µl F130 (28.3 ng/µl, 1527 bp)
8.59 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F109 + F130 (SERK-SigP + Laccase)
volume reagent
0.92 µl F109 (108.2 ng/µl, 2177 bp)
7.44 µl F130 (28.3 ng/µl, 1527 bp)
8.64 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F113 + F131 (PhyB_36AA-Linker_C-TEV + IRES)
volume reagent
0.82 µl F113 (122.2 ng/µl, 5281 bp)
2.17 µl F131 (16.4 ng/µl, 627 bp)
14.01 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F115 + F131 (TEV + IRES)
volume reagent
1.76 µl F115 (56.9 ng/µl, 2794 bp)
4.1 µl F131 (16.4 ng/µl, 627 bp)
11.14 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Negative control was prepared by replacing the volume of the insert by ddH2O.
  • The ligation was performed for 1 hour at room temperature.

Transformation of E. coli XL1-blue with ligation products F92 + F130 (IgKappa-SigP + Laccase), F109 + F130 (SERK-SigP + Laccase), F113 + F131 (PhyB_36AA-Linker_C-TEV + IRES), F115 + F131 (TEV + IRES) and negative controls and BioBrick BBa_K325909 (lucABCDEG under pBAD)

Investigator: Florian, Jeff

Aim of the experiment: Transformation of E. coli XL1-blue with ligation products F92 + F130 (IgKappa-SigP + Laccase), F109 + F130 (SERK-SigP + Laccase), F113 + F131 (PhyB_36AA-Linker_C-TEV + IRES), F115 + F131 (TEV + IRES) and negative controls and BioBrick BBa_K325909 (lucABCDEG under pBAD).


Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of QuikChange products/2 µl of plasmid DNA was added to 150 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Preparative digestion of P308 (PhyB_36AAlinker_C-TEV), P348 (Laccase RFC25) and P366 (polioviral IRES)

Investigator: Flo, Jeff

Aim of the experiment: Preparative digestion of P308 (PhyB_36AAlinker_C-TEV), P348 (Laccase RFC25) and P366 (polioviral IRES).

Procedure:

volume reagent
14 µl P308
4 µl CutSmart Buffer (10x)
1 µl SpeI-HF (20 U/µl)
1 µl PstI-HF (20 U/µl)
20 µl ddH2O
=40 µl TOTAL
volume reagent
14 µl P348
4 µl CutSmart Buffer (10x)
1 µl NgoMIV (20 U/µl)
1 µl SpeI-HF (20 U/µl)
20 µl ddH2O
=40 µl TOTAL
volume reagent
20 µl P366
4 µl CutSmart Buffer (10x)
1 µl XbaI (20 U/µl)
1 µl PstI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Reaction batches were incubated for 2.5 h at 37 °C.
  • 4 µl DNA loading buffer (10x) were added to the reaction batches after the incubation time and were loaded on a 1% agaorose gel.
  • Preparative geleletrophoresis was performed at 90 V for 60 min.

TUM13 20130624 prep Verd P308 SpeI.PstI P348 NgoMIV.SpeI P366 XbaI.PstI.png

Lane Digestion of P308 with SpeI & PstI 1 kbp DNA ladder Digestion of P348 with NgoMIV & SpeI Digestion of P366 with XbaI & PstI
result 2 further unexpected bands; seems that PstI is contaminated with a prefix restriction enzyme; used PstI enzyme batch is discareded; upper band was cut out as expected; lower band was cut out as expected; lower band was cut out

Picking of F126+F127 (NLS+ThermoNuc), AlcR QCII and npt-cassette QCII

Investigator: Jeff

Aim of the experiment: Picking of F126+F127 (NLS+ThermoNuc), AlcR QCII and npt-cassette QCII for plasmid preparation

Procedure:

  • F126+F127 (NLS+ThermoNuc) 7 clones due to high number of colonies on negative ctrl
  • AlcR QCII and npt-cassette QCII 2 clones each

Tuesday, June 25th

Sequencing of P344 pASK-Flua(Trippelmutante)

Investigator: Jeff

Aim of the experiment: Sequencing of P344. Concentration was determined by Nanodop to 132 ng/µl

Procedure:

DNA samples were prepared according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer). The samples received the following barcodes:

Label Barcode1
P344 with F83 (primer from Skerra lab! not fragment 83 from iGEM!) FR02305765

Retransformation of P35 (Viability sensor with RFP) and P249 (Thermonuc in pSB1C3)

Investigator: Jeff, Rosario

Aim of the experiment: Retransformation of P35 (Viability sensor with RFP) and P249 (Thermonuc in pSB1C3)

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • empty tube was washed with 2 µl water and the volume was added to 150 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of cell suspension were plated on chlorampenicol agar
  • The rest of the cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium; the concentrated cell suspension was plated on another chlorampenicol agar plate.

Miniprep of overnight cultures of transformated E. coli XL1 blue with QCII of P316 (npt-casette in pSB1C3), QCII of P342 (AlcR in pSB1C3) and F126+F127 (NLS in pSB1C3 + Thermonuclease)

Investigator: Louise

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with QCII of P316 (npt-casette in pSB1C3), QCII of P342 (AlcR in pSB1C3) and F126+F127 (NLS in pSB1C3 + Thermonuclease).

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
  • The resulting DNA were eluted in tubes labeled as follows:
Content New Tube
F126+F127 clone 1 P367
F126+F127 clone 2 P368
F126+F127 clone 3 P369
F126+F127 clone 4 P370
F126+F127 clone 5 P371
F126+F127 clone 6 P372
F126+F127 clone 7 P373
QCII npt clone 1 P374
QCII npt clone 2 P375
QCII AlcR clone 1 P376
QCII AlcR clone 2 P377

Analytical digestion and gelelectrophoresis of P367-P373 (NLS in pSB1C3 + Thermonuclease) with EcoRI and PstI, P374-P375 (QCII of npt casette) with XbaI and PstI, P376-P377 (QCII of AlcR) with XbaI and SpeI

Investigator: Louise, Rosario, Florian

Aim of the experiment:Analytical digestion and gelelectrophoresis of P367-P373 (NLS in pSB1C3 + Thermonuclease) with EcoRI and PstI, P374-P375 (QCII of npt casette) with XbaI and PstI, P376-P377 (QCII of AlcR) with XbaI and SpeI.

Procedure:

  • Master-Mix for P367-P373 and control P249
volume reagent
20 µl CutSmart Buffer (10x)
2.5 µl EcoRI
2.5 µl PstI
150 µl ddH2O
=175 µl TOTAL
  • Master-Mix for P374-P375 and control P316
volume reagent
8 µl CutSmart Buffer (10x)
1 µl XbaI
1 µl PstI
60 µl ddH2O
=70 µl TOTAL
  • Master-Mix for P376-P377 and control P342
volume reagent
8 µl CutSmart Buffer (10x)
1 µl XbaI
1 µl SpeI
60 µl ddH2O
=70 µl TOTAL
  • Batch for analytical digestion of P367-P377 and controls
volume reagent
17.5 µl Master-Mix
2.5 µl Plasmid DNA
=20 µl TOTAL
  • Incubation for 60 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min

TUM13 20130625 anal verd P367-P373,P249 EcoRI.PstI .png

Lane 1 kbp DNA ladder Digestion of P367 with EcoRI and PstI Digestion of P368 with EcoRI and PstI Digestion of P369 with EcoRI and PstI Digestion of P370 with EcoRI and PstI Digestion of P371 with EcoRI and PstI Digestion of P372 with EcoRI and PstI Digestion of P373 with EcoRI and PstI Digestion of P249 with EcoRI and PstI
result as expected as expected not as expected not as expected as expected as expected not as expected as expected

TUM13 20130625 anal gel QCIInpt XbaI.PstI QCIIAlcR XbaI.SpeI.png

Lane 1 kbp DNA ladder Digestion of P316 with XbaI and PstI Digestion of P374 with XbaI and PstI/SpeI Digestion of P374 with XbaI and PstI/SpeI Digestion of P375 with XbaI and PstI/SpeI Digestion of P375 with XbaI and PstI/SpeI
Digestion of P376 with XbaI and SpeI Digestion of P377 with XbaI and PstI/SpeI Digestion of P377 with XbaI and PstI/SpeI Digestion of P342 with XbaI and SpeI
result not as expected, maybe digestion with EcoRI instead of XbaI, has to be repeated not as expected, maybe digestion with EcoRI instead of XbaI, has to be repeated not as expected, maybe digestion with EcoRI instead of XbaI, has to be repeated not as expected, maybe digestion with EcoRI instead of XbaI, has to be repeated not as expected, maybe digestion with EcoRI instead of XbaI, has to be repeated
as expected not fully digested not fully digested as expected

Preparative digestion and gelelectrophoresis of P26 (Dnb1c Promotor) and P35 (RD29 Promotor) with EcoRI and KpnI

Investigator: Louise, Florian

Aim of the experiment: Preparative digestion and gelelectrophoresis of P26 (Dnb1c Promotor) and P35 (RD29 Promotor) with EcoRI and KpnI .

Procedure:

  • Batch for P26
volume reagent
20 µl Plasmid DNA
4 µl CutSmart Buffer
1 µl EcoRI
1 µl KpnI
14 µl ddH2O
=40 µl TOTAL
  • Batch for P35
volume reagent
7 µl Plasmid DNA
4 µl CutSmart Buffer
1 µl EcoRI
1 µl KpnI
27 µl ddH2O
=40 µl TOTAL


  • Incubation for 2.5 h at 37 °C.
  • 4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and the samples were loaded on a 1% agarose gel.
  • Preparative gelelectrophoresis was performed at 90 V for 60 min.

TUM13 20130625 prep Verd P26 P35 KpnI.EcoRI.png


Lane Digestion of P26 with EcoRI and KpnI 100 bp DNA ladder Digestion of P26 with EcoRI and KpnI 1 kbp DNA ladder
result not as expected, no band was cut out as expected; upper band was cut out, F132

Picking of ligation products F92 + F130 (IgKappa-SigP + Laccase), F109 + F130 (SERK-SigP + Laccase), F113 + F131 (PhyB_36AA-Linker_C-TEV + IRES), F115 + F131 (TEV + IRES) and BioBrick BBa_K325909 (lucABCDEG under pBAD) and Retransformation of P270 (IgKappa-SigP_SpyCatcher), P272 (IgKappa-SigP_SpyTag), P308 (PhyB_36AALinker_C-TEV) and P366 (IRES)

Investigator: Rosario

Aim of the experiment: Picking of ligation products F92 + F130 (IgKappa-SigP + Laccase), F109 + F130 (SERK-SigP + Laccase), F113 + F131 (PhyB_36AA-Linker_C-TEV + IRES), F115 + F131 (TEV + IRES) and BioBrick BBa_K325909 (lucABCDEG under pBAD) and Retransformation of P270 (IgKappa-SigP_SpyCatcher), P272 (IgKappa-SigP_SpyTag), P308 (PhyB_36AALinker_C-TEV) and P366 (IRES).

Procedure:

  • 2 clones were picked for each ligation product and for the biobrick
  • 1 clone was picked for each ReTrafo

Wednesday, June 26th

Miniprep of overnight cultures of transformated E. coli XL1 blue with P270, F113+F131, F109+F130, P308, F115+F131, F92+F130, F115+F131, P272, P366, lucBB

Investigator: Ingmar

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with P270, F113+F131, F109+F130, P308, F115+F131, F92+F130, F115+F131, P272, P366 and lucBB.

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
  • The resulting DNA were eluted in tubes labeled as follows:
Content New Tube
P270 Retrafo P379
F113+F131P380
F109+F130 P381
P308 Retrafo P382
F115+F131 P383
F92+F130 P384
F113+F131 P385
F115+F131 P386
F92+F130 P387
P272 Retrafo P388
P366 P389
luc BB P390
F109+F130 P391
luc BB P392

Analytical digestion and gelelectrophoresis of P374-P375 (QC II of npt-casette) and P316 (QC I of npt-casette) as a reference with XbaI and PstI

Investigator: Rosario

Aim of the experiment: Analytical digestion and gelelectrophoresis of P374-P375 (QC II of npt-casette) and P316 (QC I of npt-casette) as a reference with XbaI and PstI

Procedure:

volume reagent
2 µl CutSmart Buffer (10x)
0.25 µl XbaI
0.25 µl PstI
15 µl ddH2O
2.5 µl Plasmid DNA (P316,P374,P375)
=20 µl TOTAL
  • Incubation for 60 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min

TUM13 20130626 anal Verd P316+P374-P375 XbaI.PstI-HF.png

Lane: 1000 bp DNA ladder Digestion of P316 with XbaI and PstI Digestion of P374 with XbaI and PstI Digestion of P375 with XbaI and PstI
Result: corrupt corrupt corrupt

Picking of Retransformation of P35 (Viability sensor with RFP) and P249 (Thermonuc in pSB1C3)

Investigator: Rosario

Aim of the experiment: Picking of Retransformation of P35 (Viability sensor with RFP) and P249 (Thermonuc in pSB1C3).

Procedure:

  • 1 clone was picked for each ReTrafo

Sequencing of P367 (NLS-Thermonuclease), P376 (AlcR QC II), P374 (npt-casette QC II), P348 (Laccase), P350 (SERK-SigP_EreB_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P352 (SERK-SigP_SpyCatcher_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P354 (SERK-SigP_NanoLuc_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P356 (SERK-SigP_XylE_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P358 (SERK-SigP_SpyTag_Strep-tag-TEV-site-linker_SERK-TMD-8AAlinker_GFPmut1), P26 (DREB1C promoter)

Investigator: Rosario

Aim of the experiment: Sequencing of P367 (NLS-Thermonuclease), P376 (AlcR QC II), P374 (npt-casette QC II), P348 (Laccase), P350 (SERK-SigP_EreB_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P352 (SERK-SigP_SpyCatcher_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P354 (SERK-SigP_NanoLuc_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P356 (SERK-SigP_XylE_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P358 (SERK-SigP_SpyTag_Strep-tag-TEV-site-linker_SERK-TMD-8AAlinker_GFPmut1), P26 (DREB1C promoter).

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:


Label Barcode1 Barcode2
P367 FR02305740 (O3)
P376 FR02305741 (O3) FR02305742 (O4)
P374 FR02305743 (O3) FR02305744 (O4)
P348 FR02305745 (O3) FR02305746 (O4)
P350 FR02305747 (O3) FR02305748 (O4)
P352 FR02305749 (O3) FR02305750 (O4)
P354 FR02305751 (O3) FR02305752 (O4)
P356 FR02305753 (O3) FR02305754 (O4)
P358 FR02305755 (O3) FR02305756 (O4)
P26 FR02305757 (O4)

Thursday, June 27th

Sequencing of P387 (IgkKappa-SigP_Laccase (QCV, RFC25)), P391 (SERK-SigP_Laccase (QCV, RFC25))and P355 (SERK-SigP_XylE_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 clone 2)

Investigator: Ingmar

Aim of the experiment: Sequencing of P387 (IgkKappa-SigP_Laccase (QCV, RFC25)), P391 (SERK-SigP_Laccase (QCV, RFC25))and P355 (SERK-SigP_XylE_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 clone 2).

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:


Label Barcode1 Barcode2
P387 FR02305758 (O3) FR02305759 (O4)
P391 FR02305760 (O3)
P355 FR02305761 (O3) FR02305762 (O4)

Re-Transformation of E. coli XL1-blue with P170(SpyTag), P165(t35s), P308(PhyB_36AALinker_C-TEV), P348(Laccase QCV RFC25), P326(Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P361(N-TEV-36AAlinker-PIF6), P364(N-TEV-36AAlinker-PIF3), P266(IgKappa-SigP_Nanoluc)

Investigator: Katrin

Aim of the experiment: Re-Transformation of E. coli XL1-blue with P170(SpyTag), P165(t35s), P308(PhyB_36AALinker_C-TEV), P348(Laccase QCV RFC25), P326(Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P361(N-TEV-36AAlinker-PIF6), P364(N-TEV-36AAlinker-PIF3), P266(IgKappa-SigP_Nanoluc) Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 150 µl of competent cells were added to the tubes containing about 1 µl of plasmid DNA and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

PCR of P143 (pLuc-Actin, Amplification of pActin)

Investigator: Rosario, Flo

Aim of the experiment: PCR of P143 (pLuc-Actin, Amplification of pActin).

Procedure:

Operational sequence:

  • Reaction batch with Q5 Mastermix:
volume reagent
25 µl 2x Q5 Master Mix
2.5 µl 10 µM Forward Primer O96 (pActin_Oryza_fw)
2.5 µl 10 µM Reverse Primer O97 (pActin_Oryza_rv)
1 µl Plasmid DNA (P143)- 1:1000 dilution (desired c between 1 pg and 1 ng)
19 µL ddH2O Water
=50 µL TOTAL
  • The content has been mixed with a pipette
  • The PCR program was performed after following scheme:
Initial denaturation 98 °C 30 s
30 cycles 98 °C 10 s
62 °C 30 s
72 °C 45 s
Final extension 72 °C 2 min
Hold 4 °C infinite
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

Analytical gelelectrophoresis of F133

Investigator: Jeff

Procedure: Analytical gelelectrophoresis of F133 (PCR product of F143 with O96 & O97) to check the quality of the performed PCR.

Procedure:

  • 4.5 µl of F133 were mixed together 0.5 µl of DNA loading buffer (10x)
  • Analytical gelelectrophoresis was performed on a 1% agarose gel at 90 V for 40 min.

TUM13 20130627 anal gel F133.png

Lane: 1000 bp DNA ladder F133
Result: as expected; but byproduct visible

Analytical digestion and gelelectrophoresis of P381, P391, P347, P384, P387, P380, P385, P309, P383, P386, P390, P392

Investigator: Rosario

Aim of the experiment: Analytical digestion and gelelectrophoresis of P381, P391, P347, P384, P387, P380, P385, P309, P383, P386, P390, P392 with EcoRI and PstI

Volume Reagent
2 µl CutSmart Buffer (10x)
0.25 µl EcoRI
0.25 µl PstI
15 µl ddH2O
2.5 µl Plasmid DNA
=20 µl TOTAL
  • Incubation for 60 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min

TUM13 20130627 anal Verd P381 P391 P347 P384 P387 EcoRI.PstI.png

Lane: 1000 bp DNA ladder Digestion of P381 with EcoRI and PstI Digestion of P391 with EcoRI and PstI Digestion of P347 with EcoRI and PstI Digestion of P384 with EcoRI and PstI Digestion of P387 with EcoRI and PstI
Result: as expected as expected not fully digested as expected as expected

TUM13 20130627 anal Verd P380 P385 P309 P383 P386 P390 P392 XbaI.EcoRI.png

Lane: 1000 bp DNA ladder Digestion of P380 with EcoRI and PstI Digestion of P385 with EcoRI and PstI Digestion of P309 with EcoRI and PstI Digestion of P383 with EcoRI and PstI Digestion of P386 with EcoRI and PstI Digestion of P390 with EcoRI and PstI Digestion of P392 with EcoRI and PstI
Result: as expected as expected as expected as expected as expected as expected as expected


Preparative digestion and gelelectrophoresis of P391 (SERK-SigP_Laccase), P326 (Strep-tag-TEV-linker_SERK-TMD_8AAlinker_GFPmut1), P385 (PhyB_36AAlinker_C-TEV_IRES), P361 (N-TEV_36AAlinker_PIF6), P364 (N-TEV_36AAlinker_PIF3), P266 (IgKappa-SigP_NanoLuc), P388 (IgKappa-SigP_SpyTag), P247 (SpyCatcher), P379 (IgKappa-SigP_SpyCatcher), P374 (npt-casette) and P165 (t35S)

Investigator: Flo, Rosario

Aim of the experiment: Preparative digestion and gelelectrophoresis of P391 (SERK-SigP_Laccase), P326 (Strep-tag-TEV-linker_SERK-TMD_8AAlinker_GFPmut1), P385 (PhyB_36AAlinker_C-TEV_IRES), P361 (N-TEV_36AAlinker_PIF6), P364 (N-TEV_36AAlinker_PIF3), P266 (IgKappa-SigP_NanoLuc), P388 (IgKappa-SigP_SpyTag), P247 (SpyCatcher), P379 (IgKappa-SigP_SpyCatcher), P374 (npt-casette) and P165 (t35S).

Procedure:

Batch for preparative digestion of effectors (P328, P330, P332, P334, P336, P338) with AgeI and PstI)

volume reagent
20 µl Plasmid DNA
4 µl CutSmart Buffer (10x)
1 µl Enzyme 1 (20 U/µl)
1 µl Enzyme 2 (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
Plasmid Name Enzyme 1 Enzyme 2
P391 SERK-SigP_Laccase AgeI PstI
P326 Strep-tag-TEV-linker_SERK-TMD_8AAlinker_GFPmut1 NgoMIV PstI
P385 PhyB_36AAlinker_C-TEV_IRES SpeI PstI
P361 N-TEV_36AAlinker_PIF6 XbaI PstI
P364 N-TEV_36AAlinker_PIF3 XbaI PstI
P266 IgKappa-SigP_NanoLuc EcoRI AgeI
P388 IgKappa-SigP_SpyTag AgeI PstI
P247 SpyCatcher EcoRI NgoMIV
P379 IgKappa-SigP_SpyCatcher AgeI PstI
P374 npt-casette EcoRI XbaI
P165 t35S EcoRI SpeI
  • Incubation for 2.5 h at 37 °C.
  • 4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and they were loaded on a 1% agarose gel
  • Preparative gelelectrophoresis was performed at 90 V for 60 min.

TUM13 20130627 präp Verd P391 AgeI.PstI P326 NgoMIV.PstI P385 SpeI.PstI.png

Lane Digestion of P391 with AgeI & PstI 1kb DNA ladder Digestion of P326 with NgoMIV & PstI Digestion of P385 with SpeI & PstI
Results sideproducts?!; upper band was cut out as expected, lower band was cut out as expected

TUM13 20130627 präp Verd P361 XbaI.PstI P364 XbaI.PstI P266 EcoRI.AgeI.png

Lane Digestion of P361 with XbaI & PstI 1kb DNA ladder Digestion of P364 with XbaI & PstI Digestion of P266 with EcoRI & AgeI
Results as expected, lower band was cut out as expected, lower band was cut out as expected, lower band was cut out

TUM13 20130627 präp Verd P388 AgeI.PstI P382 NgoMIV.PstI P247 EcorI.NgoMIV.png

Lane Digestion of P388 with AgeI & PstI 1kb DNA ladder Digestion of P382 with NgoMIV & PstI Digestion of P247 with EcoRI & NgoMIV
Results as expected not expected, wrong plasmid as expected

TUM13 20130627 präp Verd P379 AgeI.PstI P374 EcoRI.XbaI P165 EcoI.SpeI.png

Lane Digestion of P379 with AgeI & PstI 1kb DNA ladder Digestion of P374 with EcoRI & XbaI Digestion of P165 with EcoRI & SpeI
Results as expected as expected as expected, but only little DNA
  • Extracted via QIAGEN gel extraction kit, digested PCR product purified via PCR Purification Kit

Ligation of F134 + F135 (SERK-SigP-Laccase + Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F136 + F137 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF6), F136 + F138 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF3) and F143 + F144 (npt-csette + t35S)

Investigator: Flo, Rosario

Aim of the experiment: Ligation of F134 + F135 (SERK-SigP-Laccase + Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F136 + F137 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF6), F136 + F138 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF3) and F143 + F144 (npt-csette + t35S).

Procedure:

  • Ligation batch for F134 + F135 (SERK-SigP-Laccase + Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1) (vector:insert = 1:3):
volume reagent
0.83 µl F134 (120.6 ng/µl, 1630 bp)
10.29 µl F135 (17.8 ng/µl, 996 bp)
5.88 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F136 + F137 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF6) (vector:insert = 1:3):
volume reagent
0.5 µl F136 (201.2 ng/µl, 5916 bp)
4.6 µl F137 (8.5 ng/µl, 771 bp)
11.9 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F136 + F138 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF3) (vector:insert = 1:3):
volume reagent
0.5 µl F136 (201.2 ng/µl, 5916 bp)
4.07 µl F138 (9.6 ng/µl, 771 bp)
12.43 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F143 + F144 (npt-csette + t35S) (vector:insert = 1:3):
volume reagent
0.53 µl F143 (188.8 ng/µl, 3587 bp)
6.05 µl F144 (3.0 ng/µl, 217 bp)
10.42 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Negative control was prepared by replacing the volume of the insert by ddH2O.
  • The ligation was performed overnight at 16 °C.

Friday, June 28th


Transformation of E. coli XL1-blue with P338(Ig-Kappa-SigP_SpyTag, Retrafo), F135+F134(SERK-SigP_Laccase+Strep-TEV_linker_SERK-TMD_linker_GFP), F136+F138(PhyB_linker_C-TEV_IRES+N-TEV_linker_PIF3), F136+F137(PhyB_linker_C-TEV_IRES+N-TEV_linker_PIF6), F143+F144(npt-Cassette+Terminator) and negative controls (F135, F136, F143)

Investigator: Katrin

Aim of the experiment: Transformation of E. coli XL1-blue with P338(Ig-Kappa-SigP_SpyTag, Retrafo), F135+F134(SERK-SigP_Laccase+Strep-TEV_linker_SERK-TMD_linker_GFP), F136+F138(PhyB_linker_C-TEV_IRES+N-TEV_linker_PIF3), F136+F137(PhyB_linker_C-TEV_IRES+N-TEV_linker_PIF6), F143+F144(npt-Cassette+Terminator) and negative controls (F135, F136, F143)

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 8 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed, for re-transformation, 1 µl of P388 was added to 150 µl of competent cells
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.


QuikChange III of AlcR (P376)

Investigator: Andi, Johanna

Aim of the experiment: QuikChange III of AlcR (P376)

Reaction batch

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid template (P376), (1:120 diluted) need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl 1:10 dilution of O84 (=10 µM)
1 µl 1:10 dilution of O85 (=10 µM)
19 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)

PCR cycling parameters

Segment Cycles Temperature Time
1 1 95 °C 30 s
2 20 95 °C 30 s
52 °C 1 min
68 °C 5 min
3 4 °C hold

Preparative digestion and gelelectrophoresis of P388 (Ig-Kappa-SigP_SpyTag), P246 (Nanoluc), F133 (PCR of pActin)

Investigator: Katrin

Aim of the experiment: Preparative digestion and gelelectrophoresis of P388 (Ig-Kappa-SigP_SpyTag), P246 (Nanoluc), F133 (PCR of pActin)

Procedure:

reaction batch for P388(Ig-Kappa-SigP_SpyTag)

volume reagent
20 µl P388
4 µl CutSmart Buffer (10x)
1 µl AgeI-HF (20 U/µl)
1 µl PstI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL

reaction batch for P246(Nanoluc)

volume reagent
20 µl P246
4 µl CutSmart Buffer (10x)
1 µl NgoMIV-HF (20 U/µl)
1 µl PstI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL


reaction batch for F133(PCR of pActin)

volume reagent
25 µl F133
5 µl CutSmart Buffer (10x)
1 µl XbaI (20 U/µl)
1 µl PstI-HF (20 U/µl)
18 µl ddH2O
=50 µl TOTAL
  • Reaction batches were incubated for 3 h at 37 °C.
  • 4 µl DNA loading buffer (10x) were added to the reaction batches after the incubation time and were loaded on a 1% agaorose gel.
  • Preparative geleletrophoresis was performed at 90 V for 60 min.

TUM13 20130628 prep Verd F133 XbaI.PstI P246 NgoMIV.PstI P388 AgeI.PstI.png

Lane Digestion of F133 with XbaI & PstI 1 kbp DNA ladder Digestion of P246 with NgoMIV,PstI Digestion of P388 with AgeI, PstI
result as expected (but smaller fragment visible), upper band was cut out as expected, lower band was cut out as expected


Ligation of F146+F147 (Ig-Kappa-SigP_SpyTag+Nanoluc) and F145+F42 (pActin+pSB1C3)

Investigator: Katrin

Aim of the experiment: Ligation of F146+F147 (Ig-Kappa-SigP_SpyTag+Nanoluc) and F145+F42 (pActin+pSB1C3)

Procedure:

  • Ligation batch for F146+F147 (Ig-Kappa-SigP_SpyTag+Nanoluc)
volume reagent
0.90 µl F146 (111.2 ng/µl, 2189 bp)
3.99 µl F147 (17.5 ng/µl, 510 bp)
12.11 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F145+F42 (pActin + pSB1C3) (batch: 30 ng vector!)
volume reagent
23.35 µl F145 (2.5 ng/µl, 1353 bp)
0.41 µl F42 (73.5 ng/µl, 2086 bp)
2.7 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=27 µl TOTAL


  • Negative controls were prepared by replacing the volume of the insert by ddH2O.
  • The ligation was performed for 1 hour at room temperature.

Picking of ReTrafos of E. coli XL1-blue with P170(SpyTag), P165(t35s), P308(PhyB_36AALinker_C-TEV), P348(Laccase QCV RFC25), P326(Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P361(N-TEV-36AAlinker-PIF6), P364(N-TEV-36AAlinker-PIF3), P266(IgKappa-SigP_Nanoluc)

Investigator: Katrin

Aim of the experiment: Picking of ReTrafos of E. coli XL1-blue with P170(SpyTag), P165(t35s), P308(PhyB_36AALinker_C-TEV), P348(Laccase QCV RFC25), P326(Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P361(N-TEV-36AAlinker-PIF6), P364(N-TEV-36AAlinker-PIF3), P266(IgKappa-SigP_Nanoluc

Procedure:

  • one clone per plate was picked
  • 4 ml LB-medium, 4 µl chloramphenicol



Dpn1 digestion and transformation of QCIII of AlcR + analytical Gel

Investigator: Katrin

Aim of the experiment: Dpn1 digestion and transformation of QCIII of AlcR + analytical Gel

Procedure:

  • 1 µl of Dpn1 (10 U/µl) was added to the reaction, the batch was incubated for one hour at 37°C
  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 8 µl of QuikChange products was added to 150 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellchloramphenicol plate.
  • 0.5 µl of loading buffer were added to 4.5 µl of Dpn1 digested plasmid
  • Electrophoresis on 1% agarose gel

TUM13 20130628 QCIII AlcR.png

Lane 1 kbp DNA ladder QCIII product of AlcR
Results as expected


Transformation of E. coli XL1-blue with ligation products F146+F147 (Ig-Kappa-SigP_SpyTag+Nanoluc) and F145+F42 (pActin+pSB1C3) and negative controls F146 and F42

Investigator: Katrin

Aim of the experiment: Transformation of E. coli XL1-blue with ligation products F146+F147 (Ig-Kappa-SigP_SpyTag+Nanoluc) and F145+F42 (pActin+pSB1C3) and negative controls F146 and F42

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 8 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

Oligohybridization of mMCSII (mMCSII_fw (O98) + mMCSII_rv (O99)) and (GGGGS)x5_TEV (GGGGSx5_TEV_fw (O102) + GGGGSx5_TEV_rv (O103))

Investigator: Jeff

Aim of the experiment: Oligohybridization of mMCSII (mMCSII_fw (O98) + mMCSII_rv (O99)) and (GGGGS)x5_TEV (GGGGSx5_TEV_fw (O102) + GGGGSx5_TEV_rv (O103)).

Procedure:

  • 25 µL of 100 pmol/µl of mMCSII_fw (O98) and mMCSII_rv (O99) were pooled and mixed together/25 µL of 100 pmol/µl of GGGGSx5_TEV_fw (O102) and GGGGSx5_TEV_rv (O103) were pooled and mixed together/
  • Heating up to 95 °C for 5 min
  • Cooling at RT in a styropor box overnight



Saturday, June 29th


Miniprep of Retrafos of P170(SpyTag), P165(t35s), P308(PhyB_36AALinker_C-TEV), P348(Laccase QCV RFC25), P326(Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P361(N-TEV-36AAlinker-PIF6), P364(N-TEV-36AAlinker-PIF3), P266(IgKappa-SigP_Nanoluc)

Investigator: Katrin

Aim of the experiment: Miniprep of Retrafos of P170(SpyTag), P165(t35s), P308(PhyB_36AALinker_C-TEV), P348(Laccase QCV RFC25), P326(Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P361(N-TEV-36AAlinker-PIF6), P364(N-TEV-36AAlinker-PIF3), P266(IgKappa-SigP_Nanoluc)

Procedure:

  • Miniprep according to QIAGEN QIAprep Kit

Transformation of E. coli XL1-blue with ligation product F145+F42 (pActin+pSB1C3, ligation overnight at 16°C) and negative control

Investigator: Katrin

Aim of the experiment: Transformation of E. coli XL1-blue with ligation products F145+F42 (pActin+pSB1C3, ligation overnight at 16°C) and negative control Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 8 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

Preparative digestion of P402 (Ig-Kappa-SigP_Nanoluc)

Investigator: Katrin

Aim of the experiment: Preparative digestion of P402 (Ig-Kappa-SigP_Nanoluc)

Procedure:

reaction batch

volume reagent
20 µl P402
4 µl CutSmart Buffer (10x)
1 µl AgeI-HF (20 U/µl)
1 µl SpeI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Reaction batches were incubated for 3 h at 37 °C.

Preparative gelelectrophoresis of F148, F149 and digestion product of P402

Investigator: Jeff

Aim of the experiment: Preparative gelelectrophoresis of F148, F149 and digestion product of P402.

Procedure:

  • Preparative gelelectrophoresis was performed on a 1% agarose gel at 90 V for 45 min.

TUM13 20130629 prep gel F148 F149 P402 AgeI.SpeI.png

Lane: 1 kbp DNA ladder F148 F149 Digestion of P402 with AgeI & SpeI
Result: as expected as expected as expected (lower band was cut out)
  • Gelpurified products were extracted via QIAquick gel extraction kit, QIAGEN.

PCR of P143 (pLuc-Actin, Amplification of pActin) and P344 (FluA R95KA45IS114T, triple mutant of FluA)

Investigator: Jeff

Aim of the experiment: PCR of P143 (pLuc-Actin, Amplification of pActin) and P344 (FluA R95KA45IS114T, triple mutant of FluA).

Procedure:

Operational sequence:

  • Reaction batch with Q5 Mastermix for P143 (pActin amplification):
volume reagent
25 µl 2x Q5 Master Mix
2.5 µl 10 µM Forward Primer O96 (pActin_Oryza_fw)
2.5 µl 10 µM Reverse Primer O97 (pActin_Oryza_rv)
1 µl Plasmid DNA (P143)- 1:1000 dilution (desired c between 1 pg and 1 ng)
19 µL ddH2O Water
=50 µL TOTAL
  • Reaction batch with Q5 Mastermix for P344 (FluA triple mutant amplification):
volume reagent
25 µl 2x Q5 Master Mix
2.5 µl 10 µM Forward Primer O104 (FluA_tripmut_fw)
2.5 µl 10 µM Reverse Primer O105 (FluA_tripmut_rv)
1 µl Plasmid DNA (P344)- 1:1000 dilution (desired c between 1 pg and 1 ng)
19 µL ddH2O Water
=50 µL TOTAL
  • The content has been mixed with a pipette
  • The PCR program was performed after following scheme for P143 (pActin amplification):
Initial denaturation 98 °C 30 s
30 cycles 98 °C 10 s
62 °C 30 s
72 °C 45 s
Final extension 72 °C 2 min
Hold 4 °C infinite
  • The PCR program was performed after following scheme for P344 (FluA triple mutant amplification)
Initial denaturation 98 °C 30 s
30 cycles 98 °C 10 s
69 °C 30 s
72 °C 16 s
Final extension 72 °C 2 min
Hold 4 °C infinite
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

Sunday, June 30th

Miniprep of F134 + F135 (SERK-SigP-Laccase + Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F136 + F137 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF6), F136 + F138 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF3), F143 + F144 (npt-csette + t35S) and ReTraFo of P388 (IgKappa-SigP_SpyTag)

Investigator: Andi

Aim of the experiment: Miniprep of F134 + F135 (SERK-SigP-Laccase + Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F136 + F137 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF6), F136 + F138 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF3), F143 + F144 (npt-csette + t35S) and ReTraFo of P388 (IgKappa-SigP_SpyTag).

Procedure:

Miniprep according to QIAGEN QIAprep Kit


Analytical digestion and gelelectrophoresis of P403 – P418

Investigator: Rosario

Aim of the experiment: Analytical digestion and gelelectrophoresis of P403 – P418.

Procedure:

  • 23x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:
volume reagent
46 µl CutSmart Buffer (10x)
5.75 µl EcoRI-HF (20 U/µl)
5.75 µl PstI-HF (20 U/µl)
345 µl ddH2O
=402.5 µl TOTAL
  • 23x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:
volume reagent
17.5 µl Mastermix for EcoRI & PstI
2.5 µl Plasmid DNA (P403 – P418)
=20 µl TOTAL
  • Analytical digestion was performed at 37 °C for 1.5 h.
  • After incubation 2.22 µl of DNA loading buffer (10x) was added to the reaction batches.
  • Analytical gelelectrophoresis was performed at 90 V for 1 h.

TUM13 20130630 analVerd P403-P410 P374 EcoRI.PstI PCRprod F153 F154.png

Lane: 1000 bp DNA ladder Digestion of P403 with EcoRI & PstI Digestion of P404 with EcoRI & PstI Digestion of P405 with EcoRI & PstI Digestion of P406 with EcoRI & PstI Digestion of P407 with EcoRI & PstI Digestion of P408 with EcoRI & PstI Digestion of P409 with EcoRI & PstI Digestion of P410 with EcoRI & PstI Digestion of P374 with EcoRI & PstI F153 F154
Result: corrupt as expected as expected as expected corrupt corrupt corrupt corrupt as expected (control) no PCR product; corrupt as expected


TUM13 20130630 analVerd P411 P412 P246 P413 P414 P391 EcoRI.PstI.png

Lane: 1000 bp DNA ladder Digestion of P411 with EcoRI & PstI Digestion of P412 with EcoRI & PstI Digestion of P246 with EcoRI & PstI (Control) Digestion of P413 with EcoRI & PstI Digestion of P414 with EcoRI & PstI Digestion of P391 with EcoRI & PstI (Control)
Result: as expected as expected as expected as expected as expected as expected


TUM13 20130630 analVerd P415-P418 P385 EcoRI.PstI.png

Lane: 1000 bp DNA ladder Digestion of P415 with EcoRI & PstI Digestion of P416 with EcoRI & PstI Digestion of P417 with EcoRI & PstI Digestion of P418 with EcoRI & PstI Digestion of P385 with EcoRI & PstI
Result: as expected as expected as expected as expected as expected (Control)

PCR of P143 (pLuc-Actin, Amplification of pActin)

Investigator: Ingmar

Aim of the experiment: PCR of P143 (pLuc-Actin, Amplification of pActin). Procedure:

Operational sequence:

  • Reaction batch with Q5 Mastermix for P143 (pActin amplification):
volume reagent
25 µl 2x Q5 Master Mix
2.5 µl 10 µM Forward Primer O96 (pActin_Oryza_fw)
2.5 µl 10 µM Reverse Primer O97 (pActin_Oryza_rv)
1 µl Plasmid DNA (P143)- 1:1000 dilution (desired c between 1 pg and 1 ng)
19 µL ddH2O Water
=50 µL TOTAL


  • The content has been mixed with a pipette
  • The PCR program was performed after following scheme for P143 (pActin amplification):
Initial denaturation 98 °C 30 s
30 cycles 98 °C 10 s
62 °C 30 s
72 °C 45 s
Final extension 72 °C 2 min
Hold 4 °C infinite
  • The PCR was performed in two different cyclers in parallel to elucidate wheather this might have an influence on the product. Furthermore one sample contained a 100 fold increased template concentration.
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
  • Analytical gelelectrophoresis was performed on a 1% agarose gel at 90 V for 60 min using NEB 2-Log DNA ladder.

TUM13 20130630 anal gel PCR P143 pActin.png

Lane: 1 kbp DNA ladder 1:1000 P143 1:1000 P143 1:10 P143
Result: length as expected, but low concentration, small amount of side products length as expected, but low concentration, small amount of side products length as expected; higher concentration but increased amount of side products, too. Fragment was labelled F155.

Preparative digestion and gelelectrophoresis of P367 (NLS+thermonuclease), P416 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6), P418 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6 ), P404 (t35S_npt-casette), P395 (SpyTag)and F154 (FluA triple mutant)

Investigator: Jeff

Aim of the experiment: Preparative digestion and gelelectrophoresis of P367 (NLS+thermonuclease), P416 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6), P418 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6 ), P404 (t35S_npt-casette), P395 (SpyTag)and F154 (FluA triple mutant).

Procedure:

Batch for preparative digestion of P404 with EcoRI and XbaI, P395 with NgoMIV and SpeI, F154 with XbaI and AgeI, P367 with XbaI and NgoMIV, P416 with SpeI and Pst I and P418 with SpeI and PstI

volume reagent
20 µl Plasmid DNA
4 µl CutSmart Buffer (10x)
1 µl Enzyme 1 (20 U/µl)
1 µl Enzyme 2 (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
volume reagent
25 µl PCR product
5 µl CutSmart Buffer (10x)
1 µl Enzyme 1 (20 U/µl)
1 µl Enzyme 2 (20 U/µl)
18 µl ddH2O
=50 µl TOTAL
Plasmid Name Enzyme 1 Enzyme 2
P404 t35S_npt-casette EcoRI XbaI
P395 SpyTag NgoMIV SpeI
P418 PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6 SpeI PstI
P367 NLS+thermonuclease XbaI NgoMIV
P416 PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6 SpeI PstI
F154 FluA triple mutant XbaI AgeI
  • Incubation for 2.5 h at 37 °C.
  • 4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and they were loaded on a 1% agarose gel
  • Preparative gelelectrophoresis was performed at 90 V for 60 min.

TUM13 20130630 prepVerd P367 XbaI.NgoMIV P416 SpeI.PstI P418 SpeI.PstI.png

Lane Digestion of P367 with XbaI & NgoMIV 1kb DNA ladder Digestion of P416 with SpeI & PstI Digestion of P418 with SpeI & PstI
Results as expected as expected as expected


TUM13 20130630 prepVerd P404 EcoRI.XbaI P395 NgoMIV.SpeI F154 XbaI.AgeI.png

Lane Digestion of P404 with EcoRI & XbaI 1kb DNA ladder Digestion of P395 with NgoMIV & SpeI Digestion of F154 with XbaI & AgeI
Results as expected as expected; lower band was extracted as expected
  • Extracted via QIAGEN gel extraction kit, digested PCR product purified via PCR Purification Kit

Ligation of F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3), F156 + F152 ((GGGGS)x5-TEV-site-linker_NLS_Thermonuclease in pSB1C3), F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3), F150 + F160 (SERK-SigP_Nanoluc_SpyTag) and F123 + F161 (FluA triple mutant in pSB1C3)

Investigator: Andi

Aim of the experiment: Ligation of F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3), F156 + F152 ((GGGGS)x5-TEV-site-linker_NLS_Thermonuclease in pSB1C3), F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3), F150 + F160 (SERK-SigP_Nanoluc_SpyTag) and F123 + F161 (FluA triple mutant in pSB1C3).

Procedure:

  • Ligation batch for F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3):
volume reagent
2.03 µl F123 (49.3 ng/µl, 2086 bp)
14.97 µl F152 (488.2 ng/µl, 102 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • F156 + F152 ((GGGGS)x5-TEV-site-linker_NLS_Thermonuclease in pSB1C3):
volume reagent
0.63 µl F156 (158.3 ng/µl, 2596 bp)
16.37 µl F152 (488.2 ng/µl, 102 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3):
volume reagent
0.67 µl F157 (148.7 ng/µl, 6711 bp)
1.71 µl F131 (16.4 ng/µl, 627 bp)
14.62 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3):
volume reagent
0.76 µl F158 (132.4 ng/µl, 6711 bp)
1.71 µl F131 (16.4 ng/µl, 627 bp)
14.59 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3):
volume reagent
1.78 µl F159 (56.1 ng/µl, 3812 bp)
15.22 µl F151 (229.4 ng/µl, 37 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • F150 + F160 (SERK-SigP_Nanoluc_SpyTag):
volume reagent
0.59 µl F150 (169.3 ng/µl, 2660 bp)
16.41 µl F160 (109 ng/µl, 39 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • F123 + F161 (FluA triple mutant in pSB1C3):
volume reagent
2.03 µl F123 (49.3 ng/µl, 2086 bp)
2.58 µl F161 (29.1 ng/µl, 522 bp)
12.39 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Week 11

Monday, July 1st

Everyday I'm swaffeling

Investigator: Le swaffleur passive

Preparative digestion and gelelectrophoresis of F155 (pActin PCR product) and P393 (Stress inducible Promoter)

Investigator: Ingmar

Aim of the experiment: Preparative digestion and gelelectrophoresis of F155 (pActin PCR product) and P393 (Stress inducible Promoter).

Procedure:

Batch for preparative digestion of F155 (pActin PCR product) with XbaI and PstI

volume reagent
28.5 µl F155
4 µl CutSmart Buffer (10x)
1 µl XbaI
1 µl PstI
5.5 µl ddH2O
=40 µl TOTAL

Batch for preparative digestion of P393 (Stress inducible Promoter)with EcoRI and SpeI"

volume reagent
20 µl P393
4 µl CutSmart Buffer (10x)
1 µl EcoRI (20 U/µl)
1 µl SpeI (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Incubation for 2.5 h at 37 °C.
  • 4.5 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and they were loaded on a 1% agarose gel
  • Preparative gelelectrophoresis was performed at 90 V for 60 min.

TUM13 20130701 prep Verd F155 P393.png

Lane 1kb DNA ladder Digestion of F155 with XbaI & PstI Digestion of P393 with EcoRI & SpeI
Results as expected, the upper band was cut out as expected, the lowest band was cut out
  • Extracted via QIAGEN gel extraction kit, digested PCR product purified via PCR Purification Kit

Ligation of F162 (pActin) + F42(pSB1C3 backbone)

Investigator: Ingmar

Aim of the experiment: Ligation of F162 (pActin) + F42(pSB1C3 backbone)

Procedure:

  • Ligation batch for ligation of F162 (pActin) + F42(pSB1C3 backbone)
volume reagent
1.36 µl F42 (100 ng)
6.64 µl F162
91 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (NEB)
=20 µl TOTAL
  • Negative controls were prepared by replacing the volume of the insert by ddH2O.
  • The ligation was performed for 1 hour at room temperature; the ligation batch was transferred to 16 °C afterwads over night.

Transformation of E. coli XL1-blue with ligation product F162+F42 (pActin+pSB1C3, ligation for 1 h at room temperature

Investigator: Ingmar

Aim of the experiment: Transformation of E. coli XL1-blue with ligation product F162+F42 (pActin+pSB1C3, ligation for 1 h at room temperature

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

Transformation of E. coli XL1-blue with ligation product F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3), F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3) and F123 + F161 (FluA triple mutant in pSB1C3)

Investigator: Rosario

Aim of the experiment: Transformation of E. coli XL1-blue with ligation product F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3), F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3) and F123 + F161 (FluA triple mutant in pSB1C3).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA were added to the tubes containing 100 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

Tuesday, July 2nd

Transformation of E. coli XL1-blue with ligation product F162+F42 (pActin+pSB1C3, ligation over night at 16 °C

Investigator: Ingmar

Aim of the experiment: Transformation of E. coli XL1-blue with ligation product F162+F42 (pActin+pSB1C3, ligation over night at 16 °C.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

Picking of F123 + F152, F157 + F131, F158 + F131, F159 + F151 and F123 + F161

Investigator: Louise

Aim of the experiment: Picking of F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3), F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3) and F123 + F161 (FluA triple mutant in pSB1C3).

Procedure:

Picked colonies were transfered into 5 ml LB-medium with 5 µl of Chloramphenicol and incubated for at 37 °C in the 180 rpm cell-culture shaker.


Ligation of F162 (pActin) + F42(pSB1C3 backbone) and of F163 (stress inducible Promoter + RFP ) + F159 (t35S-NPT-pSB1C3)

Investigator: Ingmar

Aim of the experiment: As the ligation of the prmoter done on Monday failed, it is repeated with an decreased overall volume resulting in an increased DNA concentration: Ligation of F162 (pActin) + F42(pSB1C3 backbone). Furthermore the stress inducible Promoter cloned to RFP (F163) should be ligated into a pSB1C3 in front og t35S and NPT.

Procedure:

  • Ligation batch for ligation of F162 (pActin) + F42(pSB1C3 backbone)
volume reagent
1.36 µl F42 (100 ng)
6.64 µl F162
1 µl T4 ligase buffer (10x)
1 µl T4 ligase
=10 µl TOTAL
  • The reaction was prepared twice: one time using a ligase provided by NEB and one time using a ligase providied by Thermo Scientific. Negative controls were prepared by replacing the volume of the insert by ddH2O.
  • Ligation batch for ligation of F163 (stress inducible promoter + RFP) + F159(t35S-NPT-pSB1C3 backbone)
volume reagent
1.79 µl F159 (100 ng)
8.61 µl F163
2 µl T4 ligase buffer (10x)
1 µl T4 ligase
6,6 µl H2O
=20 µl TOTAL
  • The ligation was performed for 2 hours at room temperature; the ligation batch was transferred to 16 °C afterwads over night.

Transformation of E. coli XL1-blue with ligation product F162+F42 (pActin+pSB1C3, ligation batch volume 10 µl, reaction performed at room temperature) and with F159+F163 (stress inducible promoter+RFP+t35S+NPT in pSB1C3)

Investigator: Ingmar

Aim of the experiment: Transformation of E. coli XL1-blue with ligation product F162+F42 (pActin+pSB1C3, ligation batch volume 10 µl, reaction performed at room temperature) and with F159+F163 (stress inducible promoter+RFP+t35S+NPT in pSB1C3).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

PCR of P143 (pLuc-Actin, Amplification of pActin)

Investigator: Ingmar

Aim of the experiment: PCR of P143 (pLuc-Actin, Amplification of pActin). The PCR product was phosphorylated afterwards to test blunt end cloning into a vector. Procedure:

Operational sequence:

  • Reaction batch for PCR with Q5 Mastermix for P143 (pActin amplification):
volume reagent
25 µl 2x Q5 Master Mix
2.5 µl 10 µM Forward Primer O96 (pActin_Oryza_fw)
2.5 µl 10 µM Reverse Primer O97 (pActin_Oryza_rv)
1 µl Plasmid DNA (P143)- 1:10 dilution (desired c between 1 pg and 1 ng)
19 µL ddH2O Water
=50 µL TOTAL


  • The content has been mixed with a pipette
  • The PCR program was performed after following scheme for P143 (pActin amplification):
Initial denaturation 98 °C 30 s
30 cycles 98 °C 10 s
62 °C 30 s
72 °C 45 s
Final extension 72 °C 2 min
Hold 4 °C infinite
  • The PCR was performed in two different cyclers in parallel to elucidate wheather this might have an influence on the product. Furthermore one sample contained a 100 fold increased template concentration.
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
  • Reaction batch for phosphorylation of thePCR product:
volume reagent
15 µl PCR product
2 µl 10 x forward buffer A
2 µl 10 mM ATP
1 µl Polynucleotide Kinase (Thermo Scientific)
=20 µL TOTAL
  • The reaction was performed for 1 h at 37 °C. Afterwards the mixture was inactivated by heat for 10 min at 75 °C.

Quickchange mutagenesis of P143 (Actin promoter)

Investigator: Ingmar

Aim of the experiment: Deliting the forbidden EcoRI restriction site in the promoter.

Procedure: PCR

Reaction batch 1

volume reagent
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid P143 template
0.5 µl 1:10 dilution of O106
19. 5 µl ddH2O
1 µl dNTP mix
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)

Reaction batch 2

volume reagent
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid P143 template
0.5 µl O107
19.5 µl ddH2O
1 µl dNTP mix
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment Cycles Temperature Time
1 1 95 °C 30 sec
2 15 95°C 30 sec
55°C 1 min
67°C 6,5 min
  • Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied. Furthermore 1 µl Pfu Ultra II DNA polymerase was added.
  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.


Preparative digestion and gelelectrophoresis of P352 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P358 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1) and P395 (SpyTag)

Investigator: Louise, Jeff

Aim of the experiment: Preparative digestion and gelelectrophoresis of P352 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P358 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1) and P395 (SpyTag).

  • Batch for preparative digestion of P352 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1):
volume reagent
20 µl P352
4 µl CutSmart Buffer (10x)
1 µl SpeI
1 µl PstI-HF
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P358 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1):
volume reagent
20 µl P358
4 µl CutSmart Buffer (10x)
1 µl SpeI
1 µl PstI-HF
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P395 (SpyTag):
volume reagent
20 µl P395
4 µl CutSmart Buffer (10x)
1 µl NgoMIV
1 µl SpeI
14 µl ddH2O
=40 µl TOTAL
  • Incubation at 37 °C; 2.5 h
  • 4.44 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches, digested with NgoMIV & SpeI (P395), after digestion and they were loaded on a 1% agarose gel.
  • Preparative gelelectrophoresis was performed at 90 V for 60 min.
  • Reaction batches which were digested with SpeI & PstI were purified with QIAquick PCR purification kit, QIAGEN.

TUM13 20130702 prep Verd P395 SpeI.NgoMIV.png

Lane 1kb DNA ladder Digestion of P395 with NgoMIV & SpeI
Results as expected; lower band was cut out
  • Extracted via QIAquick gel extraction kit, QIAGEN.

Ligation of F164 + F131 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3+ IRES) and F165 + F131 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES)

Investigator: Jeff

Aim of the experiment: Ligation of F164 + F131 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3+ IRES), F165 + F131 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES).

Procedure:

  • Ligation batch for F164 + F131 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3+ IRES), vector:insert = 1:3:
volume reagent
1.22 µl F164 (81.7 ng/µl, 3524 bp)
3.25 µl F131 (16.4 ng/µl, 627 bp)
12.53 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F165 + F131 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3+ IRES), vector:insert = 1:3:
volume reagent
1.65 µl F165 (60.7 ng/µl, 3224 bp)
3.56 µl F131 (16.4 ng/µl, 627 bp)
11.79 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation was performed at RT for 1 h.

Transformation of E. coli XL1-blue with ligation product F164 + F131 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3+ IRES), F165 + F131 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES) and retransformations of P352, P358 and P395

Investigator: Jeff

Aim of the experiment: Transformation of E. coli XL1-blue with ligation product F164 + F131 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3+ IRES), F165 + F131 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES) and retransformations of P352, P358 and P395.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

Ligation of F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag)

Investigator: Jeff

Aim of the experiment: Ligation of F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag).

Procedure:

  • Ligation batch for F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag), vector:insert = 1:6:
volume reagent
0.59 µl F150 (169.3 ng/µl, 2660 bp)
2.67 µl F166 (3.3 ng/µl, 39 bp)
13.79 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation was performed at 16 °C overnight.

Miniprep of E. coli XL1 blue transformed with F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3), F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3) and F123 + F161 (FluA triple mutant in pSB1C3)

Investigator: Ingmar, Jeff

Aim of the experiment: Miniprep of E. coli XL1 blue transformed with F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3), F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3) and F123 + F161 (FluA triple mutant in pSB1C3)

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN).
  • The resulting tubes were labeled as P420–P432.

Picking of of E. coli XL1 blue transformed with F152 + F156 ((GGGGS)x5-TEV-site-linker + NLS_Thermonuclease in pSB1C3)

Investigator: Jeff

Aim of the experiment: Picking of of E. coli XL1 blue transformed with F152 + F156 ((GGGGS)x5-TEV-site-linker + NLS_Thermonuclease in pSB1C3).

Procedure:

  • Picked colonies tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
  • These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overnight

Wednesday, July 3rd

Picking of of E. coli XL1 blue transformed with F162 + F42 (pActin + pSB1C3), F163 + F159 (Stress inducible promotor_RFP + t35S_NPT in pSB1C3, P395, P352, P358, F164+F131 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES), F165+F131 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES)

Investigator: Ingmar

Aim of the experiment: Picking of of E. coli XL1 blue transformed with F162 + F42 (pActin + pSB1C3), F163 + F159 (Stress inducible promotor_RFP + t35S_NPT in pSB1C3, P395, P352, P358, F164+F131 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES), F165+F131 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES).

Procedure:

  • Picked colonies tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 5 mL of LB-medium + 5 µL chloramphenicol(1000x).
  • These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overday.

Miniprep of E. coli XL1 blue transformed with F152 + F156 ((GGGGS)x5-TEV-site-linker + NLS_Thermonuclease in pSB1C3)

Investigator: Jeff

Aim of the experiment: Miniprep of E. coli XL1 blue transformed with F152 + F156 ((GGGGS)x5-TEV-site-linker + NLS_Thermonuclease in pSB1C3).

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN).
  • The resulting tubes were labeled as P433 – P435.

Transformation of E. coli XL1-blue with ligation product F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag)

Investigator: Jeff

Aim of the experiment: Transformation of E. coli XL1-blue with ligation product F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA were added to the tubes containing 100 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

Analytical digestion and gelelectrophoresis of P420 – P435

Investigator: Ingmar

Aim of the experiment: Analytical digestion and gelelectrophoresis of P420 – P435.

Procedure:

TUM13 20130703 analVerd P420 P421 F123unverdaut P422 P423 P429 P430 P431 P432 EcoRI.PstI.png

Lane: 1000 bp DNA ladder Digestion of P420 with EcoRI & PstI Digestion of P421 with EcoRI & PstI Digestion of F123 with EcoRI & PstI Digestion of P422 with EcoRI & PstI Digestion of P429 with EcoRI & PstI Digestion of P430 with EcoRI & PstI Digestion of P431 with EcoRI & PstI Digestion of P432 with EcoRI & PstI 1000 bp DNA ladder
Result: as expected as expected as expected corrupt corrupt corrupt corrupt


TUM13 20130703 analVerd P424-P428 SbfI.SpeI P404 XbaI.PstI P433-P435,P367 EcoRI.PstI.png

Lane: 1000 bp DNA ladder Digestion of P424 with EcoRI & PstI Digestion of P425 with EcoRI & PstI Digestion of P426 with EcoRI & PstI Digestion of P427 with EcoRI & PstI Digestion of P428 with EcoRI & PstI Digestion of P404 with EcoRI & PstI Digestion of P433 with EcoRI & PstI Digestion of P434 with EcoRI & PstI Digestion of P435 with EcoRI & PstI Digestion of P367 with EcoRI & PstI 1000 bp DNA ladder
Result: as expected as expected as expected corrupt corrupt corrupt corrupt as expected (control) no PCR product; corrupt as expected



Thursday, July 4th

Quickchange mutagenesis I of P444 (Actin promoter)

Investigator: Louise

Aim of the experiment: Deleting the forbidden EcoRI restriction site in the promoter.

Procedure: PCR

Reaction batch 1

volume reagent
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid P444 template
1.0 µl 1:10 dilution of O106
1.0 µl 1:10 dilution of O107
18.0 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U / µl)

Reaction batch 2

volume reagent
12.5 µl Q5 Polymerase Mastermix
1 µl Plasmid P444 template
1.25 µl 1:10 dilution of O106
1.25 µl 1:10 dilution of O107
8.0 µl ddH2O
1 µl dNTP mix 50 mM

PCR cycling parameters for batch 1

Cycles Temperature Time
1 95 °C 30 sec
20 95°C 30 sec
52°C 1 min
68°C 5 min
4°C HOLD

PCR cycling parameters for batch 2

Cycles Temperature Time
1 98 °C 30 sec
20 98°C 10 sec
55°C 30 sec
72°C 4 min
4°C HOLD
  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

The successful amplification was verified on an agerose gel and the PCR-product was transformed (by Jeff)

Ligation of F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag) and negative control F150

Investigator: Jeff, Katrin

Aim of the experiment: Ligation of F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag) and negative control F150

Procedure:

  • Ligation batch for F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag), vector:insert = 1:6:
volume reagent
0.59 µl F150 (169.3 ng/µl, 2660 bp)
2.67 µl F166 (3.3 ng/µl, 39 bp)
13.79 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation was performed at 22 °C for 1h
  • negative control was prepaed with water instead of insert F166

Test digestion of pAct in pSB1C3 (p435 to p448)

Investigator: Jeff

  • App. 500 ng of DNA from 13 different clones were digeted with EcoRI and PstI
  • Mastermix consisting of 3 µl of each enzyme 30 µl Cutsmart buffer and 120 µl water

TUM13 20130704 anal QC of pAct.gif

  • p444 and p447 were sent for sequencing

Transformation of E. coli XL1-blue with ligation product F150+F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag), F142+F147 (Ig-Kappa-SigP_SpyCatcher + Nanoluc), F139+F141 (Ig-Kappa-SigP_Nanoluc + SpyCatcher) and negative controls

Investigator: Katrin

Aim of the experiment: Transformation of E. coli XL1-blue with ligation product F150+F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag), F142+F147 (Ig-Kappa-SigP_SpyCatcher + Nanoluc), F139+F141 (Ig-Kappa-SigP_Nanoluc + SpyCatcher) and negative controls

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 8 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

Preparative digestion of P434, P412, P462, P453

Investigator: Katrin

Aim of the experiment: Preparative digestion of P434, P412, P462, P453

  • Batch for preparative digestion of P434
volume reagent
20 µl P434
4 µl CutSmart Buffer (10x)
1 µl NgoMIV
1 µl PstI-HF
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P412
volume reagent
20 µl P412
4 µl CutSmart Buffer (10x)
1 µl XbaI
1 µl PstI-HF
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P462
volume reagent
20 µl P462
4 µl CutSmart Buffer (10x)
1 µl PstI-HF
1 µl SpeI
14 µl ddH2O
=40 µl TOTAL
  • Batch for preparative digestion of P453
volume reagent
20 µl P453
4 µl CutSmart Buffer (10x)
1 µl PstI-HF
1 µl SpeI
14 µl ddH2O
=40 µl TOTAL
  • Incubation at 37 °C; 3 h, then put into freezer


Ligation of F142+F147 (IgK-SigP_Spycatcher + NanoLuc) and F141+F139 (SpyCatcher + IgK-SigP_NanoLuc)

Investigator: Louise

Aim of the experiment: Ligation of F142+F147 and F139+F141

Procedure:

  • Ligation batch for F142+F147:
volume reagent
0.81 µl F142 (123.4 ng/µl, 2489 bp)
3.51 µl F147 (17.5 ng/µl, 510 bp)
12.68 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F141+F139
volume reagent
1.83 µl F141 (54.6 ng/µl, 2425 bp)
3.43 µl F139 (21.0 ng/µl, 583 bp)
11.74 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation was performed at 22 °C for 1h
  • negative control was prepaed with water instead of inserts F147 and F139

Friday, July 5th

Ligation of F122+F169 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), F170+F168 (SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_Nanoluc) and negative controls

Investigator: Katrin

Aim of the experiment: Ligation of F122+F169 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), F170+F168 (SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_Nanoluc) and negative controls

Procedure:

  • Ligation batch for F122+F169
volume reagent
1.99 µl F122 (50.3 ng/µl, 2369 bp)
4.32 µl F169 (18.1 ng/µl, 618 bp)
10.69 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F170+F168
volume reagent
2.01 µl F170 (49.7 ng/µl, 4168 bp)
4.11 µl F168 (11.0 ng/µl, 628 bp)
10.88 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL


  • Ligation was performed at 22 °C for 1h
  • negative control was prepaed with water instead of inserts F169 and F168

Transformation of E. coli XL1-blue with ligation product F122+F169 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), F170+F168 (SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_Nanoluc) and negative controls

Investigator: Katrin

Aim of the experiment: TTransformation of E. coli XL1-blue with ligation product F122+F169 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), F170+F168 (SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_Nanoluc) and negative controls

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 8 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

Picking of E. coli XL1 blue transformed with ligation product F150+F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag), F142+F147 (Ig-Kappa-SigP_SpyCatcher + Nanoluc), F139+F141 (Ig-Kappa-SigP_Nanoluc + SpyCatcher)

Investigator: Katrin

Aim of the experiment: Picking of E. coli XL1 blue transformed with ligation product F150+F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag), F142+F147 (Ig-Kappa-SigP_SpyCatcher + Nanoluc), F139+F141 (Ig-Kappa-SigP_Nanoluc + SpyCatcher)

Procedure:

  • Picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contains 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
  • 2 colonies were picked for F150+F166 and F139+F141, 8 colonies were picked for F142+F147 (many colonies on negatibe controls)
  • These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overday.

Sequencing of P3 (pTEF2 in pTUM100), P341 (QCI of AlcR), P453 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 + IRES in pSB1C3), P462 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 + IRES in pSB1C3), P300 (Phytotchrome B)

Investigator: Rosario

Aim of the experiment: Sequencing of P3 (pTEF2 in pTUM100), P341 (QCI of AlcR), P453 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 + IRES in pSB1C3), P462 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 + IRES in pSB1C3), P300 (Phytotchrome B).

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:


Label Barcode1 Barcode2 Barcode3 Barcode4
P3 FR02233147 (O5)
P341 FR02233146 (O3) FR02233145 (O4) FR02233144 (O100) FR02233143 (O101)
P453 FR02233142 (O4)
P462 FR02233141 (O4)
P300 FR02233140 (O3) FR02233139 (O4) FR02233138 (O110) FR02233137 (O111)

Quikchange mutagenesis of P447 (Actin promoter) to remove forbidden EcoRI restriction site

Investigator: Jeff

Aim of the experiment: Quikchange mutagenesis of P447 (Actin promoter) to remove forbidden EcoRI restriction site.

Procedure:

Reaction batch 1:

volume reagent
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid P447 template (1:100)
1.0 µl 1:10 dilution of O106 (=10 µM)
1.0 µl 1:10 dilution of O107 (=10 µM)
18.0 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)


Reaction batch 2:

volume reagent
25 µl Q5 Polymerase Mastermix
1 µl Plasmid P447 template (1:100)
2.5 µl 1:10 dilution of O106 (=10 µM)
2.5 µl 1:10 dilution of O107 (=10 µM)
8.0 µl ddH2O
1 µl dNTP mix 50 mM

PCR cycling parameters for batch 1

Cycles Temperature Time
1 95 °C 30 sec
20 95°C 30 sec
52°C 1 min
68°C 4 min
1 4°C HOLD

PCR cycling parameters for batch 2

Cycles Temperature Time
1 98 °C 30 sec
20 98°C 10 sec
55°C 30 sec
72°C 2 min
3 4°C HOLD
  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

QuikChange of pYes (P3)

Investigator: Rosario

Aim of the experiment: QuikChange of pYes (P3)

Reaction batch

volume reagent Additional information
2.5 µl 10x Pfu Ultra II buffer
1 µl Plasmid template (P3), (1:10 diluted) need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl 1:10 dilution of O5 (=10 µM)
19 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)

PCR cycling parameters

Segment Cycles Temperature Time
1 1 95 °C 30 s
2 20 95 °C 30 s
52 °C 1 min
68 °C 4 min
3 4 °C hold
  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

Analytical gelelectrophoresis of the DpnI-digested QuikChange products of P447 (pActin in pSB1C3) and P3 (pTEF2 in pTUM100)

Investigator: Jeff

Procedure: Analytical gelelectrophoresis of the DpnI-digested QuikChange products of P447 (pActin in pSB1C3) and P3 (pTEF2 in pTUM100) to check whether QuikChange might be worked.

Procedure:

  • 4.5 µl of the QuikChange products of P447 (PfuUltraII Polymerase batch and Q5 Polymerase batch (see above)) and P3 (pTEF2 in pTUM100) were mixed together 0.5 µl of DNA loading buffer (10x).
  • Analytical gelelectrophoresis was performed on a 1% agarose gel at 90 V for 40 min.

TUM13 20130705 anal gel QC P3 O44.O45 P447 QCI O106.O107 .png

Lane: 2 log DNA ladder QC of P3 with O44 & O45 (PfuUltraII) QC of P447 with O106 & O107 (PfuUltraII) QC of P447 with O106 & O107 (Q5)
Result: as expected no product visible no product visible

Transformation of E. coli XL1-blue with QuikChange products of P447 (PfuUltraII and Q5) and of P3 (pTEF2 in pTUM100)

Investigator: Rosario

Aim of the experiment: Transformation of E. coli XL1-blue with QuikChange products of P447 (PfuUltraII and Q5) and of P3 (pTEF2 in pTUM100) (see above).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 8 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

Saturday, July 6th

Quikchange mutagenesis of P447 (Actin promoter) to remove forbidden EcoRI restriction site

Investigator: Le swaffeleur

Aim of the experiment: Quikchange mutagenesis of P447 (Actin promoter) to remove forbidden EcoRI restriction site.

Procedure:

Reaction batch 1:

volume reagent
2.5 µl 10x Pfu Ultra II buffer
4 µl Plasmid P447 template (1:100)
1.0 µl 1:10 dilution of O106 (=10 µM)
16.0 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)
=20 µl TOTAL


Reaction batch 2:

volume reagent
2.5 µl 10x Pfu Ultra II buffer
4 µl Plasmid P447 template (1:100)
1.0 µl 1:10 dilution of O107 (=10 µM)
16.0 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)
=20 µl TOTAL
  • The two batches were cycled seperately for the first 10 rounds.

PCR cycling parameters

Cycles Temperature Time
1 95 °C 30 sec
10 95°C 30 sec
54°C 1 min
68°C 4 min
1 4°C HOLD
  • After the ten cycles the two reaction batches were pooled together and were additionally cycled for 20 rounds with the following conditions:

PCR cycling parameters after poooling the reaction batches together:

Cycles Temperature Time
1 98 °C 30 sec
20 98°C 10 sec
54°C 30 sec
72°C 2 min
3 4°C HOLD
  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

Analytical gelelectrophoresis of QuikChange products of P447 (pActin in pSB1C3)

Investigator: Jeff

Aim of the experiment: Analytical gelelectrophoresis of QuikChange products of P447 (pActin in pSB1C3).

Procedure:

  • 9 µl of the QuikChange products were mixed with 1 µl of DNA loading buffer (10x).
  • Analytical gelelectrophoresis was performed at 90 V for 30 min on a 1% agarose gel.

TUM13 20130706 anal gel QC P447 O106.O107 withoutDMSO withDMSO.png

Lane: 2 log DNA ladder QC of P447 with O106 & O107 (PfuUltraII, without DMSO) QC of P447 with O106 & O107 (PfuUltraII, with DMSO)
Result: as expected as expected

Transformation of E. coli XL1-blue with QuikChange products of P447 (pActin in pSB1C3)

Investigator: Jeff, Le Swaffeleur

Aim of the experiment: Transformation of E. coli XL1-blue with QuikChange products of P447 (pActin in pSB1C3).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 8 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

Picking of E. coli XL1-blue transformed with ligation product F122+F169 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA) and F170+F168 (SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_NanoLuc)

Investigator: Jeff

Aim of the experiment: Picking of E. coli XL1-blue transformed with ligation product F122+F169 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA) and F170+F168 (SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_NanoLuc).

Procedure:

  • Picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contains 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
  • 2 colonies were picked for F122+F169 and F170+F168.
  • These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overnight.


Sunday, July 7th

Miniprep of E. coli XL1 blue transformed with F170+F168(SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_NanoLuc)

Investigator: Jeff, Katrin

Aim of the experiment: Miniprep of E. coli XL1 blue transformed with F170+F168(SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_NanoLuc).

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN).
  • The resulting tubes were labeled as P475 and P476.

Analytical digestion and gelelectrophoresis of P464 – P476 and P402 (control)

Investigator: Jeff, Katrin

Aim of the experiment: Analytical digestion and gelelectrophoresis of P464 – P476 and P402 (control)

  • 16x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:
volume reagent
17.5 µl Mastermix for EcoRI & PstI
2.5 µl Plasmid DNA (P464 – P476, P402)
=20 µl TOTAL
  • Analytical digestion was performed at 37 °C for 1.5 h.
  • After incubation 2  µl of DNA loading buffer (10x) were added to the reaction batches.
  • Analytical gelelectrophoresis was performed at 90 V for 1 h.


TUM13 20130707 anal verd P464-P467,P402;P475,P476 EcoRI.PstI.png

Lane: 1000 bp DNA ladder Digestion of P464 with EcoRI & PstI Digestion of P465 with EcoRI & PstI Digestion of P466 with EcoRI & PstI Digestion of P467 with EcoRI & PstI Digestion of P402 with EcoRI & PstI (control) Digestion of P475 with EcoRI & PstI Digestion of P476 with EcoRI & PstI
Result: as expected as expected as expected as expected as expected as expected as expected


TUM13 20130707 anal verd P468-P474 EcoRI.PstI.png

Lane: 1000 bp DNA ladder Digestion of P468 with EcoRI & PstI Digestion of P469 with EcoRI & PstI Digestion of P470 with EcoRI & PstI Digestion of P471 with EcoRI & PstI Digestion of P472 with EcoRI & PstI Digestion of P473 with EcoRI & PstI Digestion of P474 with EcoRI & PstI
Result: corrupt corrupt corrupt corrupt corrupt corrupt corrupt

PCR of P378 (Proteinphosphotase 1)

Investigator: Katrin, Jeff

Aim of the experiment: PCR of P378 (Proteinphosphotase 1).

Procedure:

Reaction batch for PCR with Q5 Mastermix for P378 (Proteinphosphotase 1):

volume reagent
25 µl 2x Q5 Master Mix
1 µl dNTP mix (normally not necessary!)
2.5 µl 10 µM Forward Primer O112 (PP1 for)
2.5 µl 10 µM Reverse Primer O113 (PP1 rev)
1 µl Plasmid DNA (P378) - 1:1000 dilution (desired c between 1 pg and 1 ng)
18 µL ddH2O Water
=50 µL TOTAL
  • The content has been mixed with a pipette
  • The PCR program was performed after following scheme for P378(PP1 amplification):

Cycling parameters:

Initial denaturation 98 °C 30 s
30 cycles 98 °C 10 s
62 °C 30 s
72 °C 30 s
Final extension 72 °C 2 min
Hold 4 °C infinite
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
  • The resulting product was labeled as F174.

Analytical gelelectrophoresis of F174

Investigator: Jeff

Aim of the experiment: Analytical gelelectrophoresis of F174.

Procedure:

  • 9 µl of the PCR product F174 (=PCR of P378 with O112/O113) products were mixed with 1 µl of DNA loading buffer (10x).
  • Analytical gelelectrophoresis was performed at 90 V for 60 min on a 1% agarose gel.

TUM13 20130707 anal gel F174.png

Lane: 2 log DNA ladder F174
Result: as expected

Preparative digestion and gelelectrophoresis of P422 (FluA triple mutant), P464 (IgKappa-SigP_NanoLuc_SpyCatcher) and P466 (IgKappa-SigP_NanoLuc_SpyTag)

Investigator: Katrin, Jeff

Aim of the experiment: Preparative digestion and gelelectrophoresis of P422 (FluA triple mutant), P464 (IgKappa-SigP_NanoLuc_SpyCatcher) and P466 (IgKappa-SigP_NanoLuc_SpyTag).

Procedure:

Reaction batch for P422 (FluA triple mutant):

volume reagent
20 µl P422
4 µl CutSmart Buffer (10x)
1 µl NgoMIV
1 µl SpeI
14 µl ddH2O
=40 µl TOTAL

Reaction batch for P464 (IgKappa-SigP_NanoLuc_SpyCatcher):

volume reagent
20 µl P464
4 µl CutSmart Buffer (10x)
1 µl SpeI
1 µl PstI-HF
14 µl ddH2O
=40 µl TOTAL

Reaction batch for P466 (IgKappa-SigP_NanoLuc_SpyTag):

volume reagent
20 µl P466
4 µl CutSmart Buffer (10x)
1 µl SpeI
1 µl PstI-HF
14 µl ddH2O
=40 µl TOTAL
  • Reaction batches were incubated at 37 °C for 2.5 h.
  • Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.

TUM13 20130707 prep gel P422 NgoMIV.SpeI P464 XbaI.PstI P466 XbaI.PstI.png

Lane Digestion of P422 with NgoMIV & SpeI 2 log DNA ladder Digestion of P464 with XbaI & PstI Digestion of P466 with XbaI & PstI
Results as expected; lower band was cut out as expected; lower band was cut out as expected; lower band was cut out
  • DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.

Preparative digestion of P379 (IgKappa-SigP_SpyCatcher) and F174 (Proteinphosphotase 1)

Investigator: Katrin, Jeff

Aim of the experiment: Preparative digestion of P379 (IgKappa-SigP_SpyCatcher) and F174 (Proteinphosphotase 1).

Procedure:

Reaction batch for P379 (IgKappa-SigP_SpyCatcher):

volume reagent
20 µl P379
4 µl CutSmart Buffer (10x)
1 µl AgeI-HF
1 µl PstI-HF
14 µl ddH2O
=40 µl TOTAL

Reaction batch for F174 (Proteinphosphotase 1):

volume reagent
25 µl F174
5 µl CutSmart Buffer (10x)
1 µl AgeI-HF
1 µl PstI-HF
18 µl ddH2O
=50 µl TOTAL
  • Reaction batches were incubated at 37 °C for 4 h.
  • After digestion, the product was purified with QIAquick PCR Purification Kit, Qiagen.

Ligation of F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher), F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) and F123+F175 (pSB1C3 only + PP1)

Investigator: Jeff

Aim of the experiment: Ligation of F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher), F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) and F123+F175 (pSB1C3 only + PP1).

Procedure:

Ligation batch for F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES 

in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag), vector:insert = 1:3:
volume reagent
2.01 µl F170 (49.7 ng/µl, 4168 bp)
3.50 µl F179 (12.9 ng/µl, 628 bp)
11.49 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher), vector:insert = 1:3:

volume reagent
1.65 µl F171 (60.05 ng/µl, 3868 bp)
5.80 µl F178 (12.4 ng/µl, 928 bp)
9.55 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), vector:insert = 1:3:

volume reagent
0.61 µl F176 (163.9 ng/µl, 2489 bp)
3.51 µl F147 (17.5 ng/µl, 510 bp)
12.88 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant), vector:insert = 1:3:

volume reagent
0.92 µl F109 (108.2 ng/µl, 2117 bp)
2.90 µl F177 (25.5 ng/µl, 522 bp)
13.18 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F123+F175 (pSB1C3 only + PP1), vector:insert = 1:3:

volume reagent
0.92 µl F123 (49.3 ng/µl, 2086 bp)
2.90 µl F175 (12.3 ng/µl, 972 bp)
3.59 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation was performed at 22 °C for 1 h
  • Negative controls were also prepared with water instead of insert.

Transformation of E. coli XL1-blue with ligation products F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher), F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) and F123+F175 (pSB1C3 only + PP1)

Investigator: Jeff

Aim of the experiment: Transformation of E. coli XL1-blue with ligation products F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher), F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) and F123+F175 (pSB1C3 only + PP1).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Picking of E. coli XL1 blue transformed with QuikChange products of P447 (pActin) and ligation product F123+F152 ((GGGGS)x5-TEV-cleavage-site-linker)

Investigator: Jeff

Aim of the experiment: Picking of E. coli XL1 blue transformed with QuikChange products of P447 (pActin) and ligation product F123+F152 ((GGGGS)x5-TEV-cleavage-site-linker).

Procedure:

  • Picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contains 4 mL of LB-medium + 4 µL chloramphenicol (1000x).
  • 8 colonies were picked for QuikChange products of P447 (pActin)and F139+F141 and 4 colonies were picked for ligation product F123+F152 ((GGGGS)x5-TEV-cleavage-site-linker).
  • These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overnight.

Week 12

Monday, July 8th

Analytical digestion and gelelectrophoresis of P477-P484 (pActin)

Investigator: Rosario, Ingmar

Aim of the experiment: Analytical digestion and gelelectrophoresis of P477-P484 (pActin in pSB1C3) after QC of forbidden EcoRI Restriction site.

  • 10x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:
volume reagent
15 µl Cut Smart buffer
2 µl EcoRI
2 µl PstI
30  µl ddH2O
=49 µl TOTAL
  • The volume of plasmid DNA added was calculated in order to have equal amounts of DNA of approximately 550 ng
  • Analytical digestion was performed at 37 °C for 1.5 h.
  • After incubation 2  µl of DNA loading buffer (10x) were added to the reaction batches.
  • Analytical gelelectrophoresis was performed at 90 V for 1 h.


TUM13 20130708 anal verd P477-P484 EcoRI.PstI.png

Lane: 1000 bp DNA ladder Digestion of P477 with EcoRI & PstI Digestion of P478 with EcoRI & PstI Digestion of P479 with EcoRI & PstI Digestion of P480 with EcoRI & PstI Digestion of P481 with EcoRI & PstI (control) Digestion of P482 with EcoRI & PstI Digestion of P483 with EcoRI & PstI Digestion of P484 with EcoRI & PstI
Result: as expected as expected as expected as expected as expected as expected as expected as expected
  • all quickchanges were successfull.

Quikchange mutagenesis of P477 (Actin promoter) to insert 50 bp deletion

Investigator: Rosario, Ingmar

Aim of the experiment: Quikchange mutagenesis of P477 (Actin promoter) to insert 50 bp deletion.

Procedure:

Reaction batch 1:

volume reagent
2.5 µl 10x Pfu Ultra II buffer
2,67 µl Plasmid P477 template (1:100)
1.0 µl 1:10 dilution of O108 (=10 µM)
17.330 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)
=25 µl TOTAL


Reaction batch 2:

volume reagent
2.5 µl 10x Pfu Ultra II buffer
2.67 µl Plasmid P477 template (1:100)
1.0 µl 1:10 dilution of O109 (=10 µM)
17.33 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)
=20 µl TOTAL
  • The two batches were cycled seperately for the first 10 rounds.

PCR cycling parameters

Cycles Temperature Time
1 95 °C 30 sec
10 95°C 30 sec
54°C 1 min
72°C 4.5 min
1 4°C HOLD
  • After the ten cycles the two reaction batches were pooled together and were additionally cycled for 20 rounds with the following conditions:

PCR cycling parameters after poooling the reaction batches together:

Cycles Temperature Time
1 98 °C 30 sec
20 98°C 10 sec
54°C 30 sec
72°C 2 min
3 4°C HOLD
  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.


Sequencing of P477, P486, P487, P464, P466, P476, P435

Investigator: Rosario

Aim of the experiment: Sequencing of P477 (Actin promoter), P486, P487, P464, P466, P476, P435.

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:


Label Barcode1 Barcode2
P477 FR02233136 (O3) FR02233135 (O4)
P486 FR02233134 (O3)
P487 FR02233133 (O3)
P464 FR02233132 (O4)
P466 FR02233131 (O4)
P476 FR02233130 (O3) FR02233129 (O4)
P435 FR02233128 (O3)

Preparative digestion and gelelectrophoresis of P187 (LMU GFP)

Investigator: Rosario

Aim of the experiment: Preparative digestion and gelelectrophoresis of P187 (LMU GFP).

Procedure:

Reaction batch for P187 :

volume reagent
20 µl P187
4 µl CutSmart Buffer (10x)
1 µl NgoMIV
1 µl SpeI
14 µl ddH2O
=40 µl TOTAL


  • Reaction batch was incubated at 37 °C for 2.5 h.
  • Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.

TUM13 20130708 prep Verd P187 NgoMIV.SpeI.png

Lane Digestion of P187 with NgoMIV & SpeI 2 log DNA ladder
Results as expected; lower band was cut out
  • DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.

Ligation of F109+F180 (SERK-SigP in pSB1C3 + LMU GFP) and F92+F180 (IgKappa in pSB1C3 + LMU GFP)

Investigator: Rosario

Aim of the experiment: Ligation of F109+F180 (SERK-SigP in pSB1C3 + LMU GFP) and F92+F180 (IgKappa in pSB1C3 + LMU GFP).

Procedure:

Ligation batch for F109+F180 (SERK-SigP in pSB1C3 + LMU GFP), vector:insert = 1:3:

volume reagent
4.1 µl F180 (726 bp)
0.9 µl F179 (2177 bp)
12 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

F92+F180 (IgKappa in pSB1C3 + LMU GFP), vector:insert = 1:3:

volume reagent
4.15 µl F180 (726 bp)
0.85 µl F92 (2144 bp)
12 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL


  • Ligation was performed at 22 °C for 1 h
  • Negative controls were also prepared with water instead of insert.

Picking of E. coli XL1-blue transformed with ligation products F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher), F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) and F123+F175 (pSB1C3 only + PP1))

Investigator: Rosario

Aim of the experiment: Picking of E. coli XL1-blue transformed with ligation products F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher), F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) and F123+F175 (pSB1C3 only + PP1)).

Procedure:

  • Picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contains 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
  • 2 colonies were picked for all ligation products except F176+F147 for which 4 colonies were picked.
  • These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overnight.

Tuesday, July 9th

Miniprep of E. coli XL1 blue transformed with F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F123+F175 (pSB1C3 only + PP1), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher) and F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag)

Investigator: Jeff, Florian

Aim of the experiment: Miniprep of E. coli XL1 blue transformed with F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F123+F175 (pSB1C3 only + PP1), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher) and F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag).

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN).
  • The resulting tubes were labeled as P488 to P499.

Analytical digestion and gelelectrophoresis of P488-P499 (several ligations)

Investigator: Florian

Aim of the experiment: Analytical digestion and gelelectrophoresis of P488-P499 (several ligations).

  • 15x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:
volume reagent
30 µl Cut Smart buffer
3.75 µl EcoRI
3.75 µl PstI
225  µl ddH2O
=262,5 µl TOTAL
  • 17.5 µl master mix and 2.5 µl plasmid DNA were mixed.
  • Analytical digestion was performed at 37 °C for 1 h.
  • After incubation 2  µl of DNA loading buffer (10x) were added to the reaction batches.
  • Analytical gelelectrophoresis was performed at 90 V for 1 h.


TUM13 20130709 anal Verd P488-P493 EcoRI.PstI.png

Lane: 1 kb DNA ladder Digestion of P488 F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc) with EcoRI & PstI Digestion of P489 F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc) with EcoRI & PstI Digestion of P490 F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc) with EcoRI & PstI Digestion of P491 F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc) with EcoRI & PstI Digestion of P492 F123+F175 (pSB1C3 only + PP1) with EcoRI & PstI Digestion of P493 F123+F175 (pSB1C3 only + PP1) with EcoRI & PstI
Result: as expected (950 bp) as expected (950 bp) as expected (950 bp) as expected (950 bp) as expected (972 bp) as expected (972 bp)

TUM13 20130709 anal Verd P422 P494-P499 EcoRI.PstI.png

Lane: 1 kb DNA ladder Digestion of P422 F123+F161 (FluA triple mutant + pSB1C3) with EcoRI & PstI (control) Digestion of P494 F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) with EcoRI & PstI Digestion of P495 F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) with EcoRI & PstI Digestion of P496 F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher) with EcoRI & PstI Digestion of P497 F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher) with EcoRI & PstI Digestion of P498 F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag) with EcoRI & PstI Digestion of P499 F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag) with EcoRI & PstI
Result: as expected (522 bp) strange as expected (622 bp) roughly as expected (2768 bp) roughly as expected (2768 bp) roughly as expected (2768 bp) roughly as expected (2768 bp)

Analytical gelelectrophoresis of QuikChange products of P477 (Actin promoter) to insert 50 bp deletion

Investigator: Florian

Aim of the experiment: Analytical gelelectrophoresis of QuikChange products of P477 (Actin promoter) to insert 50 bp deletion.

Procedure:

  • 9 µl of the QuikChange products were mixed with 1 µl of DNA loading buffer (10x).
  • Analytical gelelectrophoresis was performed at 90 V for 60 min on a 1% agarose gel.

TUM13 20130709 QC ins pAct.png

Lane: 1 kb DNA ladder QC of P477
Result: no product

Quikchange mutagenesis of P477 (Actin promoter) to insert 60 bp deletion, second attempt

Investigator: Jeff

Aim of the experiment: Quikchange mutagenesis of P477 (Actin promoter) to insert 60 bp deletion.

Procedure:

Reaction batch 1:

volume reagent
2.5 µl 10x Pfu Ultra II buffer
3 µl Plasmid P477 template (1:100)
1.0 µl 1:10 dilution of O108 (=10 µM)
17.0 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)
=25 µl TOTAL


Reaction batch 2:

volume reagent
2.5 µl 10x Pfu Ultra II buffer
3.0 µl Plasmid P477 template (1:100)
1.0 µl 1:10 dilution of O109 (=10 µM)
17.0 µl ddH2O
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)
=20 µl TOTAL
  • The two batches were cycled seperately for the first 25 rounds.

PCR cycling parameters

Cycles Temperature Time
1 95 °C 30 sec
10 95°C 30 sec
52°C 1 min
72°C 4.5 min
1 4°C HOLD
  • After the ten cycles the two reaction batches were pooled together, 0.5 µl of polymerase and 1 µl of dNTP-mix was added and the reaction was cycled for 25 rounds with the following conditions:

PCR cycling parameters after poooling the reaction batches together:

Cycles Temperature Time
1 98 °C 30 sec
20 98°C 10 sec
52°C 30 sec
72°C 2 min
3 4°C HOLD
  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

Wednesday, July 10th

Preparative digestion of P495 (SERK-SigP_FluA)

Investigator: Jeff

Aim of the experiment: Preparative digestion of P495 (SERK-SigP_FluA).

Procedure:

Reaction batch for P495 (SERK-SigP_FluA):

volume reagent
20 µl P495
4 µl CutSmart Buffer (10x)
1 µl AgeI-HF (20 U/µl)
1 µl PstI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • After digestion, linearized DNA was purified with QIAquick PCR purification kit, QIAGEN.

Preparative digestion and gelelectrophoresis of P486 ((GGGGS)x5-TEV-site-linker), P488 (IgKappa-SigP_SpyCatcher_NanoLuc) and P492 (Proteinphosphotase 1)

Investigator: Jeff

Aim of the experiment: Preparative digestion and gelelectrophoresis of P486 ((GGGGS)x5-TEV-site-linker), P488 (IgKappa-SigP_SpyCatcher_NanoLuc) and P492 (Proteinphosphotase 1).

Procedure:

Reaction batch for P486 ((GGGGS)x5-TEV-site-linker):

volume reagent
20 µl P486
4 µl CutSmart Buffer (10x)
1 µl XbaI (20 U/µl)
1 µl AgeI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL

Reaction batch for P488 (IgKappa-SigP_SpyCatcher_NanoLuc):

volume reagent
20 µl P488
4 µl CutSmart Buffer (10x)
1 µl XbaI (20 U/µl)
1 µl PstI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL

Reaction batch for P492 (Proteinphosphotase 1)):

volume reagent
20 µl P492
4 µl CutSmart Buffer (10x)
2 µl NgoMIV (10 U/µl)
2 µl SpeI (10 U/µl)
12 µl ddH2O
=40 µl TOTAL
  • Reaction batches were incubated at 37 °C for 2.5 h.
  • Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.

TUM13 20130710 prep gel P486 XbaI.AgeI P488 XbaI.PstI P492 NgoMIV.SpeI.png

Lane Digestion of P486 with XbaI & AgeI 2 log DNA ladder Digestion of P488 with XbaI & PstI Digestion of P492 with NgoMIV & SpeI
Results as expected; lower band was cut out as expected; lower band was cut out as expected; lower band was cut out
  • DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.

Ligation of F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1)

Investigator: Jeff

Aim of the experiment: Ligation of F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1).

Procedure:

Ligation batch for F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), vector:insert = 1:3:

volume reagent
0.63 µl F156 (158.3 ng/µl, 2596 bp)
2.88 µl F182 (4.1 ng/µl, 102 bp)
13.49 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), vector:insert = 1:3:

volume reagent
0.54 µl F181 (185.7 ng/µl, 2705 bp)
6.21 µl F135 (17.8 ng/µl, 996 bp)
10.25 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), vector:insert = 1:3:

volume reagent
1.65 µl F171 (60.5 ng/µl, 3859 bp)
1.22 µl F183 (59 ng/µl, 928 bp)
14.13 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1), vector:insert = 1:3:

volume reagent
0.92 µl F109 (108.2 ng/µl, 2117 bp)
2.44 µl F184 (54.9 ng/µl, 972 bp)
13.64 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation was performed at 16 °C overnight.
  • Negative controls were also prepared with water instead of insert.

Thursday, July 11th

Transformation of E. coli XL1-Blue with ligation products F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1)

Investigator: Jeff

Aim of the experiment: Transformation of E. coli XL1-Blue with ligation products F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA were added to the tubes containing 100 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Sequencing of P294, P296, P342, P488, P492, P495, P496 and P498

Investigator: Jeff

Aim of the experiment: Sequencing of P294, P296, P342, P488, P492, P495, P496 and P498.

Procedure:

  • The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label Barcode1 Barcode2 Barcode3
P294 FR02233072 (O100)
P296 FR02233073 (O3) FR02233074 (O4) FR02233075 (O100)
P342 FR02233076 (O3) FR02233077 (O4) FR02233078 (O100)
P488 FR02233079 (O4)
P492 FR02233080 (O3) FR02233081 (O4)
P495 FR02233082 (O3)
P496 FR02233083 (O3) FR02233084 (O4)
P498 FR02233085 (O3) FR02233086 (O4)

Friday, July 12th

QuikChange of P477 (pActin) to insert missing 60 bp

Investigator: Jeff

Aim of the experiment: QuikChange of P477 (pActin) to insert missing 60 bp.

Procedure:

Reaction batch #1, with 5% DMSO and O108 as oligonucleotide:

volume reagent
2.5 µl 10x Pfu Ultra II buffer
4 µl Plasmid P477 template (1:100)
1.0 µl 1:10 dilution of O108 (=10 µM)
1 µl dNTP mix 50 mM
1.25 µl DMSO
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)
14.75 µl ddH2O
=25 µl TOTAL

Reaction batch #2, with 5% DMSO and O109 as oligonucleotide:

volume reagent
2.5 µl 10x Pfu Ultra II buffer
4 µl Plasmid P477 template (1:100)
1.0 µl 1:10 dilution of O109 (=10 µM)
1 µl dNTP mix 50 mM
1.25 µl DMSO
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)
14.75 µl ddH2O
=25 µl TOTAL

Reaction batch #3, O108 as oligonucleotide:

volume reagent
2.5 µl 10x Pfu Ultra II buffer
4 µl Plasmid P477 template (1:100)
1.0 µl 1:10 dilution of O108 (=10 µM)
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)
16 µl ddH2O
=25 µl TOTAL

Reaction batch #4, O109 as oligonucleotide:

volume reagent
2.5 µl 10x Pfu Ultra II buffer
4 µl Plasmid P477 template (1:100)
1.0 µl 1:10 dilution of O109 (=10 µM)
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)
16 µl ddH2O
=25 µl TOTAL
  • Every reaction batch type was created twice; one batch of each type was used for the program with 55 °C as annealing temperature and one was used with 65 °C as annealing temperature.
  • The batches were cycled seperately for the first 10 rounds

PCR cycling parameters with 55 °C annealing temperature:

Cycles Temperature Time
1 95 °C 30 sec
10 95°C 30 sec
55°C 1 min
72°C 4.5 min
1 4°C HOLD

PCR cycling parameters with 65 °C annealing temperature:

Cycles Temperature Time
1 95 °C 30 sec
10 95°C 30 sec
65°C 1 min
72°C 4.5 min
1 4°C HOLD
  • After the ten cycles the reaction batches (#1 with #2 and #3 with #4) were pooled together and the reaction was cycled for further 20 rounds with the following conditions:

PCR cycling parameters with 55 °C annealing temperature:

Cycles Temperature Time
1 95 °C 30 sec
20 95°C 30 sec
55°C 1 min
72°C 4.5 min
1 4°C HOLD

PCR cycling parameters with 65 °C annealing temperature:

Cycles Temperature Time
1 95 °C 30 sec
20 95°C 30 sec
65°C 1 min
72°C 4.5 min
1 4°C HOLD
  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the pooled reaction batches and incubate for 1 h at 37 °C.

Analytical gelelectrophoresis of QuikChange products of P477 (Actin promoter) to insert 60 bp deletion

Investigator: Jeff

Aim of the experiment: Analytical gelelectrophoresis of QuikChange products of P477 (Actin promoter) to insert 60 bp deletion.

Procedure:

  • 9 µl of the QuikChange products were mixed with 1 µl of DNA loading buffer (10x).
  • Analytical gelelectrophoresis was performed at 100 V for 40 min on a 1% agarose gel.

TUM13 20130712 anal gel QCDpnI P477QCinsDMSO55C P477QCinsDMSO65C P477QCins55C P477QCins65C.png

Lane: 2 log DNA ladder QC of P477 with DMSO and annealing temperature at 55 °C QC of P477 with DMSO and annealing temperature at 65 °C QC of P477 with annealing temperature at 55 °C QC of P477 with annealing temperature at 65 °C
Result: no product no product no product no product

Picking of E. coli XL1-Blue transformed with ligation products F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1)

Investigator: Jeff

Aim of the experiment: Picking of E. coli XL1-Blue transformed with ligation products F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1).

Procedure:

  • Picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contains 4 mL of LB-medium + 4 µL chloramphenicol (1000x).
  • 2 colonies were picked for F156+F182, F171+F182, F109+F184 and 6 colonies were picked for ligation product F181+F135.
  • These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overnight.

Saturday, July 13th

Miniprep of E. coli XL1-Blue transformed with ligation products F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1)

Investigator: Jeff

Aim of the experiment: Miniprep of E. coli XL1-Blue transformed with ligation products F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1).

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
  • The resulting tubes were labeled as P508 to P519.

Analytical digestion and gelelectrophoresis of P508 – P519

Investigator: Le Swaffeleur

Aim of the experiment: Analytical digestion and gelelectrophoresis of P508 – P519.

Procedure:

  • 15x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:
volume reagent
26 µl Cut Smart buffer
3.25 µl EcoRI-HF (20 U/µl)
3.25 µl PstI-HF (20 U/µl)
195 µl ddH2O
=227.5 µl TOTAL
  • 17.5 µl master mix and 2.5 µl plasmid DNA (P508 – P519) were mixed.
  • Analytical digestion was performed at 37 °C for 1 h.
  • After incubation 2  µl of DNA loading buffer (10x) were added to the reaction batches.
  • Analytical gelelectrophoresis was performed at 90 V for 1 h.

TUM13 20130713 Analverdau P508-F519 EcoRI.PstI.png

Lane: 2 log DNA ladder P508, digested with EcoRI & PstI P509, digested with EcoRI & PstI P510, digested with EcoRI & PstI P511, digested with EcoRI & PstI P512, digested with EcoRI & PstI P513, digested with EcoRI & PstI P514, digested with EcoRI & PstI P515, digested with EcoRI & PstI P516, digested with EcoRI & PstI P517, digested with EcoRI & PstI P518, digested with EcoRI & PstI P519, digested with EcoRI & PstI 2 log DNA ladder
Result: as expected as expected as expected as expected as expected as expected as expected as expected as expected as expected as expected as expected

Sunday, July 14th

Preparative digestion and gelelectrophoresis of P428 (mMCSII_t35S_npt-casette) and P477 (pAct)

Investigator: Jeff

Aim of the experiment: Preparative digestion and gelelectrophoresis of P428 (mMCSII_t35S_npt-casette) and P477 (pAct).

Procedure:

Reaction batch for P428 (MCS-t35S-npt):

volume reagent
15 µl P428
4 µl CutSmart Buffer (10x)
1 µl EcoRI (20 U/µl)
1 µl XbaI (20 U/µl)
19 µl ddH2O
=40 µl TOTAL

Reaction batch for P477 (pAct):

volume reagent
15 µl P477
4 µl CutSmart Buffer (10x)
1 µl EcoRI (20 U/µl)
2 µl SpeI (10 U/µl)
18 µl ddH2O
=40 µl TOTAL
  • Reaction batches were incubated at 37 °C for 4 h.
  • Preparative gelelectrophoresis was performed at 90 V for 2 h in an 1% agarose gel.

TUM13 20130710 prep gel P428 Eco.XbaI P477 EcoRI.SpeI.png

Lane Digestion of P486 with XbaI & AgeI 2 log DNA ladder Digestion of P488 with XbaI & PstI 2 log DNA ladder
Results as expected; intense band was cut out as expected; lowest band was cut out
  • DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.

Ligation of F185+F186 (pAct + MCS_t35S_npt)

Investigator: Jeff

Procedure:

Ligation batch for F185+F186 (pAct + MCS_t35S_npt), vector:insert = 1:3:

volume reagent
4 µl F185 (5000 bp)
4 µl F186 (1200 bp)
1 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=10 µl TOTAL


  • Ligation was performed at 22 °C for 1 h
  • Negative controls were also prepared with water instead of insert.

Preparative digestion and gelelectrophoresis of P430 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES), P431 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES), P509 ((GGGGS)x5-TEV-site-linker_NLS_NucA) and P510 (SERK-SigP_PP1)

Investigator: Jeff

Aim of the experiment: Preparative digestion and gelelectrophoresis of P430 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES), P431 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES), P509 ((GGGGS)x5-TEV-site-linker_NLS_NucA) and P510 (SERK-SigP_PP1).

Procedure:

Reaction batch for P430 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES):

volume reagent
20 µl P430
4 µl CutSmart Buffer (10x)
2 µl SpeI (20 U/µl)
1 µl PstI-HF (20 U/µl)
13 µl ddH2O
=40 µl TOTAL

Reaction batch for P431 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES):

volume reagent
20 µl P431
4 µl CutSmart Buffer (10x)
2 µl SpeI (10 U/µl)
1 µl PstI-HF (20 U/µl)
13 µl ddH2O
=40 µl TOTAL

Reaction batch for P509 ((GGGGS)x5-TEV-site-linker_NLS_NucA):

volume reagent
20 µl P509
4 µl CutSmart Buffer (10x)
2 µl NgoMIV (10 U/µl)
1 µl PstI-HF (20 U/µl)
13 µl ddH2O
=40 µl TOTAL

Reaction batch for P510 (SERK-SigP_PP1)):

volume reagent
20 µl P510
4 µl CutSmart Buffer (10x)
1 µl AgeI-HF (20 U/µl)
1 µl PstI-HF (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Reaction batches were incubated at 37 °C for 2.5 h.
  • Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.

TUM13 20130714 prepgel P430 SpeI.PstI P431 SpeI.PstI P509 NgoMIV.PstI.png

Lane Digestion of P430 with XbaI & AgeI 2 log DNA ladder Digestion of P431 with XbaI & PstI Digestion of P509 with NgoMIV & SpeI
Results as expected as expected as expected; lower band was cut out

TUM13 20130714 prepgel P510 AgeI.PstI.png

Lane 2 log DNA ladder Digestion of P510 with AgeI & PstI
Results as expected
  • DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.

Ligation of F122+F189 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), F190+F135 (SERK-SigP_PP1 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1)

Investigator: Jeff

Aim of the experiment: Ligation of F122+F189 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), F190+F135 (SERK-SigP_PP1 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1).

Procedure:

Ligation batch for F122+F189 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), vector:insert = 1:3:

volume reagent
1.99 µl F122 (50.3 ng/µl, 2369 bp)
0.98 µl F189 (79.9 ng/µl, 618 bp)
14.03 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F190+F135 (SERK-SigP_PP1 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), vector:insert = 1:3:

volume reagent
0.45 µl F190 (224.7 ng/µl, 3155 bp)
5.32 µl F135 (17.8 ng/µl, 996 bp)
11.23 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation was performed at 16 °C overnight.
  • Negative controls were also prepared with water instead of insert.

Week 13

Monday, July 15th

Transformation of the genes to be expressend in moss (retransformation) and F122+F189 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), F190+F135 (SERK-SigP_PP1 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1)

Investigator: Jeff

Procedure for the retransformations:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 0.5 µl of DNA were added to the tubes containing 100 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • 50 µl of this cell suspension was plated on chlorampenicol plates.

Tuesday, July 16th

Preparative digestion and gelelectrophoresis of all effectors

Investigator: Jeff

Procedure:

Mastermix:

volume reagent
224 ddH2O
64 µl CutSmart Buffer (10x)
16 µl EcoRI-HF (20 U/µl)
16 µl PstI-HF (20 U/µl)
  • 20 µl of DNA were mixed with 20 µl of Mastermix and were incubated at 37 °C for 4 h.
  • Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.

TUM13 20130716 prepgel 1.png

Lane 2 log DNA ladder Digestion of P20 (GFP) with EcoRI & PstI 2 log DNA ladder Digestion of P196 (XylE) with EcoRI & PstI 2 log DNA ladder Digestion of P245 (EreB) with EcoRI & PstI
Results as expected as expected as expected as expected as expected as expected

TUM13 20130716 prepgel 2.jpg

Lane 2 log DNA ladder Digestion of P246 (nLuc) with EcoRI & PstI 2 log DNA ladder Digestion of P330 (SERK-SigP_NLuc) with EcoRI & PstI 2 log DNA ladder Digestion of P346 (Dehydrochlorinase) with EcoRI & PstI
Results as expected as expected as expected as expected as expected as expected


TUM13 20130716 prepgel 3.jpg
The digestion of P350 was separated for an additional hour because there might be contaminations of the vector that is smaller than the protein-coding BioBrick we want to express. The separated bands looked like that:
TUM13 20130716 prepgel 3b.jpg

Lane 2 log DNA ladder Digestion of P350 (EreB-Receptor) with EcoRI & PstI 2 log DNA ladder Digestion of P354 (nLuc-Receptor) with EcoRI & PstI 2 log DNA ladder Digestion of P402 (IgK-nLuc) with EcoRI & PstI
Results as expected as expected as expected as expected as expected as expected


TUM13 20130716 prepgel 4.jpg

Lane 2 log DNA ladder Digestion of P476 (SpyCatcher-Receptor_IRES_IgK_SpyTag-nLuc) with EcoRI & PstI 2 log DNA ladder Digestion of P496 (SpyTag-Receptor_IRES_nLuc_SpyCatcher with EcoRI & PstI 2 log DNA ladder Digestion of P498 (SpyCatcher-Receptor_IRES_IgK_nLuc-SpyTAG) with EcoRI & PstI
Results as expected as expected as expected as expected as expected as expected


TUM13 20130716 prepgel 5.jpg TUM13 20130716 prepgel 5b.jpg

Lane 2 log DNA ladder Digestion of P502 (IgK-GFP) with EcoRI & PstI 2 log DNA ladder Digestion of P512 (SpyTAG-Receptor-IRES_SpyCatcher-nLuc) with EcoRI & PstI 2 log DNA ladder Digestion of P516 (FluA-Recetpro) with EcoRI & PstI
Results as expected as expected as expected as expected as expected as expected


  • DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.

Miniprep of P520 to P525: P520 (Bba_KK509000,Cauliflower Mosaic Virus 35S promoter) P521-525 (pAct(with_del)-MCS-T35S-npt

Investigator: Jeff

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
  • The resulting tubes were labeled as P520 to P525.

Analytical digestion and gelelectrophoresis of P520 - P525

Investigator: Jeff

Procedure:

  • 7x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:
volume reagent
7 µl Cut Smart buffer
1.75 µl EcoRI-HF (20 U/µl)
1.75 µl PstI-HF (20 U/µl)
38.5 µl ddH2O
=49 µl TOTAL
  • 7 µl master mix and 3 µl plasmid DNA were mixed.
  • Analytical digestion was performed at 37 °C for 1.5bsp;h.
  • After incubation 2  µl of DNA loading buffer (10x) were added to the reaction batches.
  • Analytical gelelectrophoresis was performed at 90 V for 1 h.

TUM13 20130713 Analverdau P520-25 EoRI.PstI.jpg

Lane: 2 log DNA ladder P428, dsted with EcoRI & PstI P522, digested with EcoRI & PstI P522, digested with EcoRI & PstI P523, digested with EcoRI & PstI P524, digested with EcoRI & PstI P525, digested with EcoRI & PstI
Result: control no insert ;( no insert ;( no insert ;( no insert ;( no insert ;( no insert ;(

Wednesday, July 17th

Thursday, July 18th

Analytical digestion and gelelectrophoresis of P537 – P540 and analytical gelelectrophoresis of F206 and F207

Investigator: Jeff

Aim of the experiment: Analytical digestion and gelelectrophoresis of P537 – P540 and analytical gelelectrophoresis of F206 and F207.

Procedure:

volume reagent
2.5 µl Plasmid DNA (P537 – P540)
2 µl Cut Smart buffer
0.25 µl EcoRI-HF
0.25 µl PstI-HF
15 µl ddH2O
=20 µl TOTAL
  • Analytical digestion was performed at 37 °C for 1.5 h.
  • After incubation 2  µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.
  • Analytical gelelectrophoresis was performed at 90 V for 1 h.

TUM13 20130718 analgel P537-P540 EcoRI.PstI F206 F207.png

Lane: 2 log DNA ladder Digestion of P537 F122+F189 (SERK-SigP_SERK-TMD in pSB1C3 + (GGGGS)x5-TEV-site-linker_NLS_NucA) with EcoRI & PstI Digestion of P538 F122+F189 (SERK-SigP_SERK-TMD in pSB1C3 + (GGGGS)x5-TEV-site-linker_NLS_NucA) with EcoRI & PstI Digestion of P539 F190+F135 (SERK-SigP_PP1 in pSB1C3 only + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1) with EcoRI & PstI Digestion of P540 F190+F135 (SERK-SigP_PP1 in pSB1C3 only + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1) with EcoRI & PstI F206 F207
Result: as expected (916 bp) as expected (916 bp) as expected (2080 bp) as expected (2080 bp) as expected (3484 bp) as expected (3484 bp)

PCR to insert 60 missing base-pairs into pActin (new method)

Investigator: Jeff

Aim of the experiment: PCR to insert 60 missing base-pairs into pActin (new method)

Procedure:

Reaction batch for PCR with Q5 Mastermixes for P477 (pActin):

volume reagent
25 µl 2x Q5 Master Mix
2.5 µl 10 µM Forward Primer O114 (pAct_ins_fw)
2.5 µl 10 µM Reverse Primer O115 (pAct_ins_rv)
1 µl Plasmid DNA (P477) - 1:1000 dilution (desired c between 1 pg and 1 ng)
19 µL ddH2O Water
=50 µL TOTAL
volume reagent
25 µl 2x Q5 Master Mix
2.5 µl 10 µM Forward Primer O114 (pAct_ins_fw)
2.5 µl 10 µM Reverse Primer O115 (pAct_ins_rv)
1 µl Plasmid DNA (P477) - 1:100 dilution (desired c between 1 pg and 1 ng)
19 µL ddH2O Water
=50 µL TOTAL
  • The content has been mixed with a pipette
  • The PCR program was performed after following scheme:

Cycling parameters:

Initial denaturation 98 °C 30 s
30 cycles 98 °C 10 s
72 °C 30 s
72 °C 2 min
Final extension 72 °C 2 min
Hold 4 °C infinite
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
  • The resulting product was labeled as F206 for the 1:1000 batch and F207 for the 1:100 batch.

Friday, July 19th

Saturday, July 20th

Sunday, July 21st

Week 14

Monday, July 22nd

Arrival of the correct pActin 5 from Physcomitrella patens from Reski lab

This plasmid was named p541

Tuesday, July 23rd

Wednesday, July 24th

PCR of P541 (PppActin5)

Investigator: Jeff

Aim of the experiment: PCR of P541 (PppActin5).

Procedure:

Reaction batch for PCR with Q5 Mastermix for P541 (PppActin5):

volume reagent
25 µl 2x Q5 Master Mix
1 µl dNTP mix (normally not necessary!)
2.5 µl 10 µM Forward Primer O116 (PppAct_f)
2.5 µl 10 µM Reverse Primer O117 (PppAct_r)
1 µl Plasmid DNA (P541) - 1:1000 dilution (desired c between 1 pg and 1 ng)
18 µL ddH2O Water
=50 µL TOTAL
  • The content has been mixed with a pipette
  • The PCR program was performed after following scheme:

Cycling parameters:

Initial denaturation 98 °C 30 s
30 cycles 98 °C 10 s
53 °C 30 s
72 °C 30 s
Final extension 72 °C 2 min
Hold 4 °C infinite
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
  • The resulting product was labeled as F208.

Analytical gelelectrophoresis of F208

Investigator: Jeff

Aim of the experiment: Analytical gelelectrophoresis of F208.

Procedure:

  • 5 µl of the PCR product F208 (=PCR of P541 with O116/O117) products were mixed with 2 µl of DNA loading buffer (10x).
  • Analytical gelelectrophoresis was performed at 90 V for 60 min on a 1% agarose gel.

TUM13 20130724 anal gel F208.jpg

Lane: 2 log DNA ladder F208
Result: as expected

Preparative digestion of P208 (PCR of PppActin5)

Investigator: Jeff


Procedure:

Reaction batch for F208 (PCR of PppActin5):

volume reagent
25 µl F208
5 µl CutSmart Buffer (10x)
1 µl EcoRI-HF
1 µl SpeI-HF
18 µl ddH2O
=45 µl TOTAL


  • Reaction batch was incubated at 37 °C for 2 h.
  • After digestion, the product was purified with QIAquick PCR Purification Kit, Qiagen.


Ligation and transformation of F209 + F41 (PppActin5 in pSB1C3)

Investigator: Jeff


Procedure:

Ligation batch for F209 (PppActin5), vector:insert = 1:3:

volume reagent
7 µl F209 (36.9 ng/µl, ??? bp)
1 µl F189 (89.9 ng/µl, ??? bp)
1 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=10 µl TOTAL


  • Ligation was performed at RT for 1 hour.
  • Negative controls were also prepared with water instead of insert.

The ligation products were transformed into E. coli XL-1 Blue.

Thursday, July 25th

Picking of E. coli XL1 blue transformed with ligation product of F208 and F41 (PppActin5 in pSB1C3)

Investigator: Jeff

Procedure:

  • Picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contains 4 mL of LB-medium + 4 µL chloramphenicol (1000x).
  • 6 colonies were picked for ligation of F208 and F41 (PppActin5 in pSB1C3).
  • These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overnight.

Friday, July 26th

Miniprep of P542 to P547 (pSB1C2_PppActin5)

Investigator: Jeff

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
  • The resulting tubes were labeled as P542 to P547.

Analytical digestion and gelelectrophoresis of P542 – P547

Investigator: Jeff

Procedure:

volume reagent
3 µl Plasmid DNA (P542 – P547)
1 µl Cut Smart buffer
0.25 µl EcoRI-HF
0.25 µl PstI-HF
5.5 µl ddH2O
=10 µl TOTAL
  • Analytical digestion was performed at 37 °C for 1.5 h.
  • After incubation 2  µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.
  • Analytical gelelectrophoresis was performed at 90 V for 1 h.

TUM13 20130726 analgel P542-P547 EcoRI.PstI.jpg

Lane: 2 log DNA ladder Digestion of P542 (PppActin5) with EcoRI & PstI Digestion of P543 (PppActin5) with EcoRI & PstI Digestion of P544 (PppActin5) with EcoRI & PstI Digestion of P545 (PppActin5) with EcoRI & PstI Digestion of P546 (PppActin5) with EcoRI & PstI Digestion of P547 (PppActin5) with EcoRI & PstI
Result: result unclear because insert and backbone have the same length! result unclear because insert and backbone have the same length! result unclear because insert and backbone have the same length! result unclear because insert and backbone have the same length! result unclear because insert and backbone have the same length! result unclear because insert and backbone have the same length!

Unfortunately the correctness of the clones could not be seen in this experiment. Thus a second digestion was performed:


volume reagent
3 µl Plasmid DNA (P542 – P547)
1 µl Cut Smart buffer
0.25 µl EcoRI-HF
0.25 µl PstI-HF
0.25 µl NcoI (form Fermentas
5.25 µl ddH2O
=10 µl TOTAL
  • Analytical digestion was performed at 37 °C for 1.5 h.
  • After incubation 2  µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.
  • Analytical gelelectrophoresis was performed at 90 V for 1 h.

TUM13 20130726 analgel P542-P547 EcoRI.PstI.NcoI.jpg

Lane: 2 log DNA ladder Digestion of P542 (PppActin5) with EcoRI & PstI Digestion of P543 (PppActin5) with EcoRI & PstI Digestion of P544 (PppActin5) with EcoRI & PstI Digestion of P545 (PppActin5) with EcoRI & PstI Digestion of P546 (PppActin5) with EcoRI & PstI Digestion of P547 (PppActin5) with EcoRI & PstI
Result: Clone correct: backbone and PppActin5 Clone wrong: only backbone, no insert Clone correct: backbone and PppActin5 Clone correct: backbone and PppActin5 Clone wrong: only backbone, no insert Clone correct: backbone and PppActin5


Sequencing of P544 and P545

Investigator: Ingmar


Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:


Label Barcode1 Barcode2
P544 FR02233095 FR02233087
P545 FR02233093 FR02233094

Saturday, July 27th

Sunday, July 28st

Week 15

Monday, July 29th

QuikChange of P544 (PppActin5)

Investigator: Jeff

Procedure:

volume reagent
2.5 µl 10x Pfu Ultra II buffer
4 µl Plasmid P544 template (1:100)
1.0 µl 1:10 dilution of O118 (=10 µM)
1.0 µl 1:10 dilution of O119 (=10 µM)
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)
15 µl ddH2O
=25 µl TOTAL
volume reagent
2.5 µl 10x Pfu Ultra II buffer
4 µl Plasmid P544 template (1:100)
1.0 µl 1:10 dilution of O120 (=10 µM)
1.0 µl 1:10 dilution of O121 (=10 µM)
1 µl dNTP mix 50 mM
0.5 µl Pfu Ultra II DNA polymerase (2.5 U/µl)
15 µl ddH2O
=25 µl TOTAL

PCR cycling parameters with 55 °C annealing temperature:

Cycles Temperature Time
1 95 °C 30 sec
10 95°C 30 sec
55°C 1 min
72°C 5 min
1 4°C HOLD
  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the pooled reaction batches and incubate for 1 h at 37 °C.

Tuesday, July 30th

Wednesday, July 31st

Preparative digestion and gelelectrophoresis of P537 (SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA)

Investigator: Jeff

Aim of the experiment: Preparative digestion and gelelectrophoresis of P537 (SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA).

Procedure:

Reaction batch for P537 (SERK-SigP_SERK-TMD_(GGGGS)-TEV-site-linker_NLS_NucA):

volume reagent
20 µl P537
4 µl CutSmart Buffer (10x)
2 µl XbaI (20 U/µl)
2 µl PstI-HF (20 U/µl)
12 µl ddH2O
=40 µl TOTAL
  • Reaction batches were incubated at 37 °C for 2 h.
  • Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.

TUM13 20130731 prepgel P537 XbaI.PstI.png

Lane 2 log DNA ladder Digestion of P537 with XbaI & PstI
Results as expected; lower band was cut out
  • DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.
  • Resulting fragment was named as F210.

Ligation of F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA)

Investigator: Jeff

Aim of the experiment: Ligation of F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA).

Procedure:

Ligation batch for F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA, vector:insert = 1:3:

volume reagent
0.53 µl F187 (189.9 ng/µl, 7346 bp)
0.68 µl F210 (55.3 ng/µl, 916 bp)
15.79 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA, vector:insert = 1:3:

volume reagent
0.73 µl F188 (136.7 ng/µl, 7346 bp)
0.68 µl F210 (55.3 ng/µl, 916 bp)
15.59 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation was performed at 16 °C overnight.
  • Negative controls were also prepared with water instead of insert.

Thursday, August 1st

Transformation of E. coli XL1-Blue with BBa_K864100 (SYFP2), BBa_E0020 (eCFP) and ligation products F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA)

Investigator: Jeff

Aim of the experiment: Transformation of E. coli XL1-Blue with BBa_K864100 (SYFP2), BBa_E0020 (eCFP) and ligation products F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA were added to the tubes containing 100 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Friday, August 2nd

Picking of E. coli XL1-Blue transformed with BBa_K864100 (SYFP2), BBa_E0020 (eCFP) and ligation products F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA)

Investigator: Jeff

Aim of the experiment: Picking of E. coli XL1-Blue transformed with BBa_K864100 (SYFP2), BBa_E0020 (eCFP) and ligation products F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA).

Procedure:

  • Picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contains 4 mL of LB-medium + 4 µL chloramphenicol (1000x).
  • 2 colonies were picked for BBa_K864100, BBa_E0020, F187+F210 and F188+F210 each.
  • These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overnight.

Saturday, August 3rd

Miniprep of E. coli Xl1-Blue transformed with BBa_K864100 (SYFP2), BBa_E0020 (eCFP) and ligation products F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA)

Investigator: Jeff

Aim of the experiment: Miniprep of E. coli Xl1-Blue transformed with BBa_K864100 (SYFP2), BBa_E0020 (eCFP) and ligation products F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA).

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
  • The resulting tubes were labeled as P558 to P565.

Analytical digestion and gelelectrophoresis of P558 – P565

Investigator: Jeff

Aim of the experiment: Analytical digestion and gelelectrophoresis of P558 – P565.

Procedure:

Mastermix for analytical digestion with EcoRI and PstI

volume reagent
20 µl Cut Smart buffer
2.5 µl EcoRI-HF
2.5 µl PstI-HF
150 µl ddH2O
=175 µl TOTAL
  • 17.5 µl of the mastermix were mixed together with 2.5 µl of Plasmid DNA (P558 – P565)
  • Analytical digestion was performed at 37 °C for 1.5 h.
  • After incubation 2  µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.
  • Analytical gelelectrophoresis was performed at 90 V for 1 h.

TUM13 20130803 analgel P558-P561 EcoRI.PstI.png

Lane: Digestion of P558 (BBa_K864100) Digestion of P559 (BBa_K864100) 2 log DNA ladder Digestion of P560 (BBa_E0020) Digestion of P561 (BBa_E0020)
Result: as expected (723 bp) as expected (723 bp) as expected (723 bp) as expected (723 bp)

TUM13 20130803 analgel P562-P565 EcoRI.PstI.png

Lane: Digestion of P562 (F187+F210) Digestion of P563 (F187+F210) 2 log DNA ladder Digestion of P564 (F188+F210) Digestion of P565 (F188+F210)
Result: as expected (6191 bp) as expected (6191 bp) not as expected; corrupt (6191 bp) as expected (6191 bp)

Sunday, August 4th

Week 16

Monday, August 5th

Preparative digestion and gelelectrophoresis of P540 (SERK-SigP_PP1_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P558 (SYFP2), P560 (eCFP), P562 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES_SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and P565 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES_SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA)

Investigator: Jeff

Aim of the experiment: Preparative digestion and gelelectrophoresis of P540 (SERK-SigP_PP1_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P558 (SYFP2), P560 (eCFP), P562 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES_SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and P565 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES_SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA).

Procedure:

Reaction batch for P540:

volume reagent
20 µl P540
4 µl CutSmart Buffer (10x)
2 µl EcoRI-HF (20 U/µl)
2 µl PstI-HF (20 U/µl)
4 µl XhoI (10 U/µl)
8 µl ddH2O
=40 µl TOTAL

Reaction batch for P558, P560, P562, P565:

volume reagent
20 µl P558, P560, P562, P565
4 µl CutSmart Buffer (10x)
2 µl EcoRI-HF (20 U/µl)
2 µl PstI-HF (20 U/µl)
12 µl ddH2O
=40 µl TOTAL
  • Reaction batches were incubated at 37 °C for 2 h.
  • Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.

TUM13 20130805 prepgel P540 EcoRI.PstI.XhoI P558 EcoRI.PstI P560 EcoRI.PstI.png

Lane Digestion of P540 with EcoRI & PstI & XhoI 2 log DNA ladder Digestion of P558 with EcoRI & PstI Digestion of P560 with EcoRI & PstI
Results as expected; upper band was cut out as expected; lower band was cut out as expected; lower band was cut out

TUM13 20130805 prepgel P562 EcoRI.PstI P565 EcoRI.PstI.png

Lane Digestion of P562 with EcoRI & PstI 2 log DNA ladder Digestion of P565 with EcoRI & PstI
Results as expected; upper band was cut out as expected; upper band was cut out
  • DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.
  • Resulting fragment was named as F214 – F218.

PCR of P558 (SYFP2) and P560 (eCFP) to introduce RFC25 pre- and suffixes

Investigator: Jeff

Aim of the experiment: PCR of P558 (SYFP2) and P560 (eCFP) to introduce RFC25 pre- and suffixes. Procedure:

Reaction batch for PCR with Q5 Mastermixes for P558 (SYFP2):

volume reagent
25 µl 2x Q5 Master Mix
2.5 µl 10 µM Forward Primer O122 (SYFP2_fw)
2.5 µl 10 µM Reverse Primer O123 (SYFP2_rv)
1 µl Plasmid DNA (P558) - 1:100 dilution (desired c between 1 pg and 1 ng)
19 µL ddH2O Water
=50 µL TOTAL

Reaction batch for PCR with Q5 Mastermixes for P560 (eCFP):

volume reagent
25 µl 2x Q5 Master Mix
2.5 µl 10 µM Forward Primer O124 (eCFP_fw)
2.5 µl 10 µM Reverse Primer O125 (eCFP_rv)
1 µl Plasmid DNA (P560) - 1:100 dilution (desired c between 1 pg and 1 ng)
19 µL ddH2O Water
=50 µL TOTAL
  • The content has been created on ice and mixed with a pipette
  • The gradient PCR program was performed after following scheme with following conditions (Tm=67 °C; ΔG=2 °C; P558 in row 1(=65.0 °C); P560 in row 12 (=69.2 °C)):

Cycling parameters:

Initial denaturation 98 °C 30 s
30 cycles 98 °C 10 s
Tm=67 °C; ΔG=2 °C 30 s
72 °C 22 s
Final extension 72 °C 2 min
Hold 4 °C infinite
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
  • The resulting product was labeled as F212 for the the PCR products of P558 and F213 for the PCR product of P560.

Analytical gelelectrophoresis of F212 (SYFP2 RFC25) and F213 (eCFP RFC25)

Investigator: Jeff

Aim of the experiment: Analytical gelelectrophoresis of F212 (SYFP2 RFC25) and F213 (eCFP RFC25).

Procedure:

  • 4.5 µl of the PCR product F212 and F213 products were mixed with 0.5 µl of DNA loading buffer (10x).
  • Analytical gelelectrophoresis was performed at 90 V for 60 min on an 1% agarose gel.

TUM13 20130805 analgel F211 F212.png

Lane: F212 2 log DNA ladder F213
Result: as expected as expected

Tuesday, August 6th

Oligohybridization of O128(G-TEV-site-G_fw) and O129 (G-TEV-site-G_rv)

Investigator: Jeff

Aim of the experiment: Oligohybridization of O128(G-TEV-site-G_fw) and O129 (G-TEV-site-G_rv).

Procedure:

  • 25 µL of O128 (100 µM) and 25 µL of O129 (100 µM) were pooled together.
  • Heating up to 95 °C for 5 min.
  • Cooling at RT in a styropor box overnight.

Preparative digestion of F212 (SYFP2 RFC25) and F213 (eCFP RFC25)

Investigator: Jeff

Aim of the experiment: Preparative digestion of F212 (SYFP2 RFC25) and F213 (eCFP RFC25).

Procedure:

Reaction batch for F212:

volume reagent
25 µl F212
5 µl CutSmart Buffer (10x)
1 µl XbaI (20 U/µl)
1 µl AgeI-HF (20 U/µl)
18 µl ddH2O
=50 µl TOTAL

Reaction batch for F213:

volume reagent
25 µl F213
5 µl CutSmart Buffer (10x)
1 µl XbaI (20 U/µl)
1 µl AgeI-HF (20 U/µl)
18 µl ddH2O
=50 µl TOTAL
  • Reaction batches were incubated at 37 °C for 2 h.
  • Digested products were purified with QIAquick PCR Purification Kit, QIAGEN.

Ligation of F123+F219 (pSB1C3 + SYFP2) and F123+F220 (eCFP)

Investigator: Jeff

Aim of the experiment: Ligation of F123+F219 (pSB1C3 + SYFP2) and F123+F220 (eCFP).

Procedure:

Ligation batch for F123+F219 (pSB1C3 + SYFP2), vector:insert = 1:3:

volume reagent
1 µl F123 (49.3 ng/µl, 2086 bp)
0.4 µl F219 (114.9 ng/µl, 723 bp)
15.6 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F123+F220 (pSB1C3 + eCFP), vector:insert = 1:3:

volume reagent
1 µl F123 (49.3 ng/µl, 2086 bp)
1.2 µl F220 (41.7 ng/µl, 723 bp)
14.8 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation was performed at 16 °C overnight.


Analytical digestion and gelelectrophoresis of further clones

Investigator: Jeff


Procedure: The plasmids were cut with 0.2 µl of EcoRI and SpeI in a volume of 10 µl.

  • Analytical digestion was performed at 37 °C for 2 h.
  • After incubation 3  µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.
  • Analytical gelelectrophoresis was performed at 90 V for 1 h.

TUM13 20130806 analgel PppAct5mMCST35Snpt EcoRI.SpeI-.jpg

Lane: 2log DNA ladder clone 1 clone 2 clone 3 clone 4 clone 5 clone 6 clone 7 clone 8
Result: wrong result wrong result wrong result wrong result wrong result wrong result wrong result wrong result



Preparative digestion of P428 (mMCS-T35S-npt) and P554 (PppAct5-QCII)

Investigator: Jeff


Procedure:

Reaction batch for P428:

volume reagent
20 µl P428
5 µl CutSmart Buffer (10x)
1 µl EcoRI (20 U/µl)
1 µl XbaI (20 U/µl)
23 µl ddH2O
=50 µl TOTAL

Reaction batch for P554:

volume reagent
20 µl F213
5 µl CutSmart Buffer (10x)
1 µl EcoRI (20 U/µl)
2 µl SpeI (10 U/µl)
2 µl NcoI (10 U/µl)
18 µl ddH2O
=50 µl TOTAL
  • Reaction batches were incubated at 37 °C for 2 h.

TUM13 20130806 prepgel PPPAct5diezwote.jpg

Lane 2 log DNA ladder Digestion of P428 with EcoRI & XbaI Digestion of P554 with EcoRI & SpeI
Results as expected; highest band was cut out as expected; highest band was cut out

The bands were extracted from the gel and termed F221 and F222. The concentrations were determined to 42 and 37 ng/µl.


Ligation of F221+F222 (PppAct5 + pSB1C3-mMCS-T35S-npt)

Investigator: Jeff


Procedure:

Ligation batch for F221+F222 (PppAct5 + pSB1C3-mMCS-T35S-npt), vector:insert = 1:3:

volume reagent
5 µl F221 (42 ng/µl, 3700 bp)
11 µl F222 (37 ng/µl, 2000 bp)
2 µl T4 ligase buffer (10x)
2 µl T4 ligase (1 U/µl)
=20 µl TOTAL


  • Ligation was performed at room temperature overnight.


Transformation of E. coli XL1-Blue with ligation F221+F222

Investigator: Jeff

Procedure:

  • After 1 h of ligation 10 µl were used for transformation.
  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA were added to the tubes containing 100 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Wednesday, August 7th

Transformation of E. coli XL1-Blue with ligation F221+F222

Investigator: Jeff

Procedure:

  • After 1 h of ligation 10 µl were used for transformation.
  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA were added to the tubes containing 100 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.


Picking of of E. coli XL1 blue with PppAct5-mMCS-T35S-npt from the ligation performed after 1 h

Investigator: Jeff

Procedure:

  • PppAct5-mMCS-T35S-npt: Colonies were picked from chloramphenicol plates.
  • Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
  • 12 colonies were picked.
  • These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight

Thursday, August 8th

Analytical digestion and gelelectrophoresis of further clones

Investigator: Jeff


Procedure: The plasmids were cut with 0.2 µl of EcoRI and SpeI in a volume of 10 µl.

  • Analytical digestion was performed at 37 °C for 2 h.
  • After incubation 3  µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.
  • Analytical gelelectrophoresis was performed at 90 V for 1 h.

TUM13 20130806 analgel PppAct5mMCST35Snpt EcoRI.SpeI.jpg

Lane: 2log DNA ladder clone 1 clone 2 clone 3 clone 4 clone 5 clone 6 clone 7 clone 8 clone 9 clone 10
Result: wrong result wrong result wrong result wrong result wrong result wrong result wrong result wrong result wrong result wrong result

All these clones were discarded.
Additionally the fragments for the ligation were controlled.

Lane: 2log DNA ladder F185 F211 F221 F222
Result: correct correct correct correct

Picking of of E. coli XL1 blue with PppAct5-mMCS-T35S-npt from the ligation performed over night

Investigator: Jeff

Procedure:

  • PppAct5-mMCS-T35S-npt: Colonies were picked from chloramphenicol plates.
  • Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
  • 40 colonies were picked.
  • These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight

Friday, August 9th

Miniprep of E. coli Xl1-Blue transformed with ligation of PppAct5 into mMCS-T35S-npt

Investigator: Jeff


Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
  • The resulting tubes were labeled as P574 to P613.

Analytical digestion and gelelectrophoresis of P574 – P613

Investigator: Jeff

Aim of the experiment: Analytical digestion and gelelectrophoresis of P574 – P613.

Procedure:

Mastermix for analytical digestion with EcoRI

volume reagent
45 µl Cut Smart buffer
8 µl EcoRI-HF
400 µl ddH2O
=453 µl TOTAL
  • 9 µl of the mastermix were mixed together with 1 µl of Plasmid DNA
  • Analytical digestion was performed at 37 °C for 2 h.
  • After incubation 3  µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.
  • Analytical gelelectrophoresis was performed at 90 V for 1 h.

TUM13 20130809 analgel PppAct5mMCST35Snpt Gel1 EcoRI.jpg

Lane: 2log DNA ladder clone 1 clone 2 clone 3 clone 4 clone 5 clone 6 clone 7 clone 8 clone 9 clone 10
Result: wrong result wrong result wrong result wrong result wrong result wrong result wrong result wrong result wrong result good clone => only faint additional band

TUM13 20130809 analgel PppAct5mMCST35Snpt Gel2 EcoRI.jpg

Lane: 2log DNA ladder clone 1 clone 2 clone 3 clone 4 clone 5 clone 6 clone 7 clone 8 clone 9 clone 10
Result: wrong result wrong result wrong result wrong result wrong result wrong result wrong result best clone => no additional band (best clone) wrong result wrong result

TUM13 20130809 analgel PppAct5mMCST35Snpt Gel3 EcoRI.jpg

Lane: 2log DNA ladder clone 1 clone 2 clone 3 clone 4 clone 5 clone 6 clone 7 clone 8 clone 9 clone 10
Result: wrong result wrong result wrong result wrong result wrong result wrong result wrong result wrong result correct size, but additional bands correct size, but additional bands

TUM13 20130809 analgel PppAct5mMCST35Snpt Gel4 EcoRI.jpg

Lane: 2log DNA ladder clone 1 clone 2 clone 3 clone 4 clone 5 clone 6 clone 7 clone 8 clone 9 clone 10
Result: wrong result wrong result correct size, but additional bands wrong result wrong result correct size, but additional bands wrong result wrong result wrong result wrong result

Saturday, August 10th

Miniprep of E. coli Xl1-Blue transformed with ligation products F123+F219 (SYFP RFC25) and F123+F220 (eCFP RFC25)

Investigator: Jeff

Aim of the experiment: Miniprep of E. coli Xl1-Blue transformed with ligation products F123+F219 (SYFP RFC25) and F123+F220 (eCFP RFC25).

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
  • The resulting tubes were labeled as P614 to P617.

Analytical digestion and gelelectrophoresis of P614 – P617

Investigator: Jeff

Aim of the experiment: Analytical digestion and gelelectrophoresis of P614 – P617.

Procedure:

Mastermix for analytical digestion with EcoRI and PstI

volume reagent
2.5 µl Plasmid DNA (P614 – P617)
2 µl Cut Smart buffer
0.25 µl EcoRI-HF
0.25nbsp;µl PstI-HF
15 µl ddH2O
=20 µl TOTAL
  • Analytical digestion was performed at 37 °C for 1 h.
  • After incubation 2  µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.
  • Analytical gelelectrophoresis was performed at 120 V for 30 min.

TUM13 20130810 analgel P614-P617 EcoRI.PstI.png

Lane: 2 log DNA ladder Digestion of P614 (SYFP2 RFC25) Digestion of P615 (SYFP2 RFC25) Digestion of P616 (eCFP RFC25) Digestion of P617 (eCFP RFC25)
Result: as expected (723 bp) as expected (723 bp) as expected (723 bp) as expected (723 bp)

Sunday, August 11th

Preparative digestion of P615 (SYFP2 RFC25)

Investigator: Jeff

Aim of the experiment: Preparative digestion of P615 (SYFP2 RFC25)

Procedure:

Preparative digestion and gelelectrophoresis of P617 (eCFP RFC25)

Investigator: Jeff

Aim of the experiment: Preparative digestion of P617 (eCFP RFC25)

Procedure:

Ligation of F224+F223 (creation of Gly-TEV-site-Gly_SYFP2)

Investigator: Jeff

Aim of the experiment: Ligation of F224+F223 (creation of Gly-TEV-site-Gly_SYFP2).

Procedure:

Ligation batch for F224+F223 (SYFP2 + Gly-TEV-site-Gly), vector:insert = 1:3:

volume reagent
0.66 µl F224 (152.1 ng/µl, 2086 bp)
0.34 µl F223 (2506.9 ng/µl, 35 bp)
16 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation was performed at 16 °C overnight.

Miniprep of Retrafos of P516, P330, P245, P496, P346, P196, P20, P476, P554, P498, P246, P354, P428, P512, P402, P197, P350, P502

Investigator: Louise, Christopher

Aim of the experiment: Miniprep of Retrafos of P516, P330, P245, P496, P346, P196, P20, P476, P554, P498, P246, P354, P428, P512, P402, P197, P350, P502.

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
  • The new plasmids were numbered P618 - P635 (see Inventory)

Week 17

Monday, August 12th

Sequencing of P509, P512, P516, P537, P540, P562, P565, P583, P591

Investigator: Louise

Aim of the experiment: Sequencing of P509, P512, P516, P537, P540, P562, P565, P583, P591

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label Barcode1 (fw) Barcode2 (rev)
P509 FR02233103
P632 FR02233102 FR02233101
P633 FR02233100 FR02233099
P537 FR02233098 FR02233097
P540 FR02233096 FR02233104
P562 FR02233105 FR02233106
P565 FR02233107 FR02233108
P583 FR02233109 FR02233110 (Primer O131)
P591 FR02233111 FR02233088 (Primer O131)


Transformation of E. coli XL1-Blue with P337,P540, P583 and P591

Investigator: Johanna

Aim of the experiment: Retransformation of E. coli XL1-Blue with P337,P540, P583 and P591

Procedure:

  • 1,5 µl of dd H2O were added to the empty tubes
  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 1,5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Transformation of E. coli XL1-Blue with ligation product F224+F223 (SYFP2 + G-TEV-site-G) and negative control of F224

Investigator: Louise

Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product F224+F223 (SYFP2 + G-TEV-site-G) and negative control of F224

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 1,5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Tuesday, August 13th

Gelextraction and Ligation of digested P583 (PppActin5-MCS-T35S-npt) and P591 (PppActin5-MCS-T35S-npt)

Investigator: Louise

Aim of the experiment: Gelextraction and selfligation to verify correct cut of digested P583 (PppActin5-MCS-T35S-npt) and P591 (PppActin5-MCS-T35S-npt)

Procedure:

  • After preparative digestion of P583 and P591 with SpfI and MfeI the gel extraction was performed after manufacturer`s protocol (QIAquick Gel Extraction Kit, QIAGEN)
  • the extracted fragments were labelled F226 (digested P583) and F227 (digested P591)
  • ligation was performed without extra inserts with just 100 ng of cut plasmid to test if it was cut as expected
  • Ligation batch for self-ligation of F226
volume reagent
0.6 µl F226 (164.1 ng/µl)
16.4 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for self-ligation of F227
volume reagent
0.4 µl F227 (243.4 ng/µl)
16.6 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL


Transformation of E. coli XL1-Blue with ligation product of self-ligated F226 and F227 and with Biobrick BBa_J45014

Investigator: Louise

Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product of self-ligated F226 and F227 and with Biobrick BBa_245014

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 1,5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Picking of Retrafos of P337, P540, P583 and P591 and of Ligation F224 + F223

Investigator: Jeff

Aim of the experiment: Picking of Retrafos of P337 (SERK-SigP_XylE), P540 (SERK-SigP_PP1_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P583 (PppActin5-MCS-T35S-npt) and P591 (PppActin5-MCS-T35S-npt) and of Ligation F224 + F223 (SYFP2 RFC25 in pSB1C3 + Oligohybridization of G-TEV-site-G)

Procedure:

  • Retrafos 1 clone each, Ligation 4 clones due to approx. 50 % positive neg. ctrl
  • 4 ml LB medium w/ 4 µl chloramphenicol
  • Incubation overnight at 37 °C in the 180 rpm

Wednesday, August 14th

Picking of of E. coli XL1 blue transformed with BBa_J45014 (banana odour)

Investigator: Louise

Aim of the experiment: Picking of of E. coli XL1 blue transformed with BBa_J45014 (banana odour)

Procedure:

  • BBa_J45014 (banana odour): 4 Colonies were picked from chloramphenicol plates.
  • Each colony was transferred into a tube containing 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
  • These tubes were transferred into a cell culture shaker at 37 °C and were incubated overnight

Analytical digestion and gelelectrophoresis of P640-P643 (G-TEV-site-G_SYFP2) and P615 with EcoRI and PstI and P637, P639 with EcoRI and MfeI (old and new)

Investigator: Louise, Johanna

Aim of the experiment: Analytical digestion and gelelectrophoresis of P640-P643 (G-TEV-site-G_SYFP2) and P615 (SYFP2, RFC25) with EcoRI and PstI and P637 (PppActin5-MCS-T35S-npt), P639 (PppActin5-MCS-T35S-npt) with EcoRI and MfeI (old and new)

Procedure:

  • batch for digestion of P640-P643
volume reagent
8.5 µl Plasmid DNA
2 µl Cut Smart buffer
0.25 µl EcoRI-HF
0.25 µl PstI-HF
9 µl ddH2O
=20 µl TOTAL
  • batch for digestion of P615
volume reagent
2.5 µl Plasmid DNA
2 µl Cut Smart buffer
0.25 µl EcoRI-HF
0.25 µl PstI-HF
15 µl ddH2O
=20 µl TOTAL
  • batch for digestion of P637 and P639
volume reagent
8.5 µl Plasmid DNA
2 µl Cut Smart buffer
0.25 µl EcoRI-HF
0.25 µl MfeI (old or new)
9 µl ddH2O
=20 µl TOTAL
  • Analytical digestion was performed at 37 °C for 1.5 h.
  • After incubation 2  µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.
  • Analytical gelelectrophoresis was performed at 90 V for 1 h.

TUM13 20130814 anal Verd P615 EcoRI.PstI P637 P639 EcoRI.MfeI.alt.neu P640-P643 EcoRI.PstI.png

Lane: DNA ladder Digestion of P615 (SYFP2 RFC25-reference) Digestion of P637 (with old MfeI) Digestion of P637 (with new MfeI) Digestion of P639 (with old MfeI) Digestion of P639 (with new MfeI) Digestion of P640 Digestion of P641 Digestion of P642 Digestion of P643
Result: as expected corrupt MfeI as expected corrupt MfeI as expected as expected as expected as expected as expected

Sequencing of P614, P617, P637, P639 and P641

Investigator: Flo, Johanna

Aim of the experiment: Sequencing of P614, P617, P637, P639 and P641

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label Barcode Primer
P614 FR02233112 03
P617 FR02233113 03
P641 FR02233114 03
P637 FR02233119 061
P639 FR02233115 061

Miniprep of Retrafos of P337, P583, P540, P591 and of ligation product F224+F223

Investigator: Johanna

Aim of the experiment: Miniprep of Retrafos of P337, P583, P540, P591 and of ligation product F224+F223

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
  • The new plasmids were numbered as followed:
Plasmid Miniprep-Plasmid
P337 P636
P583 P637
P540 P638
P591 P639
F224+223 (version 1) P640
F224+223 (version 2) P641
F224+223 (version 3) P642
F224+223 (version 4) P643

Miniprep of Retrafos of banana odour, P583 and P591

Investigator: Florian

Aim of the experiment: Miniprep of Retrafos of banana odour, P583 and P591.

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
  • The new plasmids were numbered as followed:
Plasmid Miniprep-Plasmid
Banana 1 P644
Banana 2 P645
Banana 3 P646
Banana 4 P647
P583 P648
P591 P649

Preparative digestion of P644 (banana odour), P648 & P649 (PppAct5-mMCS-T35S-npt)

Investigator: Florian


Procedure:

Reaction batch for P644:

volume reagent
20 µl P644
5 µl CutSmart Buffer (10x)
1 µl EcoRI (20 U/µl)
1 µl PstI (20 U/µl)
23 µl ddH2O
=50 µl TOTAL

Reaction batch for P648:

volume reagent
40 µl P648
10 µl CutSmart Buffer (10x)
2 µl MfeI (20 U/µl)
0 µl was empty :'( SbfI (20 U/µl)
46 µl ddH2O
=50 µl TOTAL

Reaction batch for P649:

volume reagent
40 µl P649
10 µl CutSmart Buffer (10x)
2 µl MfeI (20 U/µl)
2 µl SbfI (20 U/µl)
46 µl ddH2O
=50 µl TOTAL
  • Reaction batches were incubated at 37 °C for 3 h.

TUM13 20130806 prepgel PPPAct5diezwote.jpg

Lane 2 log DNA ladder Digestion of P428 with EcoRI & XbaI Digestion of P554 with EcoRI & SpeI
Results as expected; highest band was cut out as expected; highest band was cut out

The bands were extracted from the gel and termed F221 and F222. The concentrations were determined to 42 and 37 ng/µl.

Thursday, August 15th

Preparative digestion of P414 and P640 + Preparative Gelelectrophoresis + Gelextraction

Investigator: Andi


Procedure:

Reaction batch for P414:

volume reagent
20 µl P414
4 µl CutSmart Buffer (10x)
1 µl EcoRI (20 U/µl)
1 µl PstI (20 U/µl)
14 µl ddH2O
=40 µl TOTAL


Reaction batch for P640:

volume reagent
20 µl P640
4 µl CutSmart Buffer (10x)
1 µl EcoRI (20 U/µl)
2 µl NgoMIV (20 U/µl)
13 µl ddH2O
=40 µl TOTAL
  • Reaction batches were incubated at 37 °C for 2.5 h.
  • Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.

TUM13 20131508 praep verdau P414 EcoRI PstI P640 EcoRI NgoMIV.png

Lane Digestion of P414 with EcoRI & PstI 1kB DNA ladder Digestion of P640 with EcoRI & NgoMIV
Results as expected; upper band was cut out as expected; lower band was cut out
  • Gelextraction was performed after preparative Gelelectrophoresis
  • The resulting fragments were named F231 (Laccase) + F232

Ligation of F230 with F191, F192, F193, F194, F195, F196, F197, F198, F199, F200, F201, F202, F203, F204, F205, F214, F217 and F218

Investigator: Louise, Florian

Aim of the experiment: Ligation of F230 with F191, F192, F193, F194, F195, F196, F197, F198, F199, F200, F201, F202, F203, F204, F205, F214, F217 and F218

Procedure:

Ligation batch for F230+F191 (plasmid with mini MCS + GFP cytoplasmatic), vector:insert = 1:3

volume reagent
0.41 µl F230 (243.7 ng/µl, 5796 bp)
18.7 µl F191 (2 ng/µl, 723 bp)
0.0 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL


Ligation batch for F230+F192 (plasmid with mini MCS + catecholdioxygenase cytoplasmatic), vector:insert = 1:3

volume reagent
0.41 µl F230 (243.7 ng/µl, 5796 bp)
5.94 µl F192 (8 ng/µl, 918 bp)
10.65 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F230+F193 (plasmid with mini MCS + EreB cytoplasmatic), vector:insert = 1:3

volume reagent
0.41 µl F230 (243.7 ng/µl, 5796 bp)
1.85 µl F193 (35 ng/µl, 1254 bp)
14.74 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F230+F194 (plasmid with mini MCS + Nanoluciferase cytoplasmatic), vector:insert = 1:3

volume reagent
0.41 µl F230 (243.7 ng/µl, 5796 bp)
8.80 µl F194 (3 ng/µl, 510 bp)
7.79 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F230+F195 (plasmid with mini MCS + nanoluciferase secretory_SERK), vector:insert = 1:3

volume reagent
0.41 µl F230 (243.7 ng/µl, 5796 bp)
3.99 µl F195 (8 ng/µl, 616 bp)
12.60 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F230+F196 (plasmid with mini MCS + gutathiontransferase cytoplasmatic), vector:insert = 1:3

volume reagent
0.41 µl F230 (243.7 ng/µl, 5796 bp)
1.17 µl F196 (28 ng/µl, 633 bp)
15.42 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F230+F197 (plasmid with mini MCS + EreB membrane associated), vector:insert = 1:3

volume reagent
0.41 µl F230 (243.7 ng/µl, 5796 bp)
11.12 µl F197 (11 ng/µl, 2362 bp)
5.47 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F230+F198 (plasmid with mini MCS + Nanoluc membrane associated), vector:insert = 1:3

volume reagent
0.41 µl F230 (243.7 ng/µl, 5796 bp)
6.44 µl F198 (13 ng/µl, 1618 bp)
10.15 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL


Ligation batch for F230+F199 (plasmid with mini MCS + nanoluc secretory_Ig Kappa), vector:insert = 1:3

volume reagent
0.41 µl F230 (243.7 ng/µl, 5796 bp)
1.16 µl F199 (26 ng/µl, 583 bp)
15.43 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F230+F200 (plasmid with mini MCS + Nanoluc+IRES+Spytag_Nanoluc), vector:insert = 1:3

volume reagent
0.41 µl F230 (243.7 ng/µl, 5796 bp)
3.00 µl F200 (47 ng/µl, 2725 bp)
13.59 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F230+F201 (plasmid with mini MCS + Nanoluc+IRES+Nanoluc_Spycatcher), vector:insert = 1:3

volume reagent
0.41 µl F230 (243.7 ng/µl, 5796 bp)
3.81 µl F201 (37 ng/µl, 2725 bp)
12.78 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F230+F202 (plasmid with mini MCS + Nanoluc+IRES+Nanoluc_Spytag), vector:insert = 1:3

volume reagent
0.41 µl F230 (243.7 ng/µl, 5796 bp)
2.66 µl F202 (53 ng/µl, 2725 bp)
13.93 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F230+F203 (plasmid with mini MCS + IgKappa_GFP secretory), vector:insert = 1:3

volume reagent
0.41 µl F230 (243.7 ng/µl, 5796 bp)
1.09 µl F203 (38 ng/µl, 800 bp)
15.50 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F230+F204 (plasmid with mini MCS + Nanoluc+IRES+Spycatcher_Nanoluc), vector:insert = 1:3

volume reagent
0.41 µl F230 (243.7 ng/µl, 5796 bp)
2.17 µl F204 (65 ng/µl, 2725 bp)
14.42 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL


Ligation batch for F230+F205 (plasmid with mini MCS + FluA membrane associated), vector:insert = 1:3

volume reagent
0.41 µl F230 (243.7 ng/µl, 5796 bp)
2.11 µl F205 (40 ng/µl, 1630 bp)
14.48 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F230+F214 (plasmid with mini MCS + PP1 membrane associated), vector:insert = 1:3

volume reagent
0.41 µl F230 (243.7 ng/µl, 5796 bp)
0.95 µl F214 (113 ng/µl, 2080 bp)
15.64 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F230+F217 (plasmid with mini MCS + PIF3 safety), vector:insert = 1:3

volume reagent
0.41 µl F230 (243.7 ng/µl, 5796 bp)
2.76 µl F217 (116 ng/µl, 6191 bp)
13.83 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F230+F217 (plasmid with mini MCS + PIF3 safety), vector:insert = 3:1

volume reagent
1.15 µl F230 (243.7 ng/µl, 5796 bp)
0.86 µl F217 (116 ng/µl, 6191 bp)
14.99 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F230+F218 (plasmid with mini MCS + PIF6 safety), vector:insert = 1:3

volume reagent
0.41 µl F230 (243.7 ng/µl, 5796 bp)
2.50 µl F218 (128 ng/µl, 6191 bp)
14.09 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F230+F218 (plasmid with mini MCS + PIF3 safety), vector:insert = 3:1

volume reagent
1.15 µl F230 (243.7 ng/µl, 5796 bp)
0.78 µl F218 (116 ng/µl, 6191 bp)
15.07 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation was performed at roomtemperature, 1 h

Ligation of F230 with F231

Investigator: Louise, Christopher

Aim of the experiment: Ligation of F230 with F231

Procedure:

Ligation batch for F230+F231 (plasmid with mini MCS + Laccase membrane-associated), vector:insert = 1:3

volume reagent
0.41 µl F230 (243.7 ng/µl, 5796 bp)
14.33 µl F231 (9.5 ng/µl, ~2630 bp)
2.26 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation was performed at room temperature, 1 h

Transformation of E. coli XL1-Blue with ligation products F230+F191, F230+F192, F230+F193, F230+F194, F230+F195, F230+F196, F230+F197, F230+F198, F230+F199, F230+F200, F230+F201, F230+F202, F230+F203, F230+F204, F230+F205, F230+F214, F230+F217, F230+F218, F217+F230, F218+F230 and negative control of F230

Investigator: Louise, Christopher

Aim of the experiment: Transformation of E. coli XL1-Blue with ligation products F230+F191, F230+F192, F230+F193, F230+F194, F230+F195, F230+F196, F230+F197, F230+F198, F230+F199, F230+F200, F230+F201, F230+F202, F230+F203, F230+F204, F230+F205, F230+F214, F230+F217, F230+F218, F217+F230, F218+F230 and negative control of F230

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 1,5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.


Friday, August 16th

Sequencing of P640 (SYFP2 RFC25 in pSB1C3 + Oligohybridization of G-TEV-site-G)

Investigator: Rosario

Aim of the experiment: Sequencing of P640.

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label Barcode Primer
P640 FR02233116 03

Preparative digestion of P640 (SYFP2 RFC25 in pSB1C3 + Oligohybridization of G-TEV-site-G) with EcoRI+NgoMIV

Investigators: Louise, Jeff

Aim of the experiment: Preparative digestion of P640 (SYFP2 RFC25 in pSB1C3 + Oligohybridization of G-TEV-site-G) with EcoRI+NgoMIV

Procedure:

volume reagent
33 µl P640
4 µl CutSmart Buffer (10x)
2 µl NgoMIV (10 U/µl)
1 µl EcoRI Hf (20 U/µl)
=40 µl TOTAL
  • Reaction batches were incubated at 37 °C for 3 h.

20130816 prepgel P640 EcoRI.NgoMIV.png

Lane 2 log DNA ladder Digestion of P640 with EcoRI & NgoMIV
Results as expected; band was cut out

The band was extracted from the gel via QIAGEN Gel Extraction Kit and termed F233. The concentration was 31.1 ng/µl.


Transformation of E. coli XL1-Blue with ligation product F230+F231(plasmid with mMCS+membrane-bound Laccase) and negative control of F230, Retrafo of P541 (PppActin5) and P449(stress inducible Promoter_RFP +t35S-NPT-pSB1C3)

Investigator: Rosario

Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product F230+F231(plasmid with mMCS+membrane-bound Laccase) and negative control of F230, Retrafo of P541 (PppActin5) and P449(stress inducible Promoter_RFP +t35S-NPT-pSB1C3).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 1,5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Ligation of F233+F225 (creation of eCFP_Gly-TEV-site-Gly_SYFP2)

Investigator: Jeff

Aim of the experiment: Creation of eCFP_Gly-TEV-site-Gly_SYFP2

Procedure:

Ligation batch, vector:insert = 1:3

volume reagent
3.22 µl F233 (31.1 ng/µl, 2834 bp)
1.93 µl F225 (39.6 ng/µl, 723 bp)
11.85 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • neg. ctrl. w/ water instead of insert
  • Ligation was performed at room temperature, 1 h

Miniprep of E. coli XL1-Blue transformed with ligation products F230+F191, F230+F192, F230+F193, F230+F194, F230+F195, F230+F196, F230+F197, F230+F198, F230+F199, F230+F200, F230+F201, F230+F202, F230+F203, F230+F204, F230+F205, F230+F214, F230+F217, F217+F230, F230+F218, F218+F230, F230+F228

Investigator: Jeff, Rosario, Louise, Christopher

Aim of the experiment: Miniprep of E. coli XL1-Blue transformed with ligation products F230+F191, F230+F192, F230+F193, F230+F194, F230+F195, F230+F196, F230+F197, F230+F198, F230+F199, F230+F200, F230+F201, F230+F202, F230+F203, F230+F204, F230+F205, F230+F214, F230+F217, F217+F230, F230+F218, F218+F230, F230+F228

Procedure:

  • Ligation products were labelled B to V (in the same order as in Aim of the experiment, above)
  • Two clones for every ligation product except for H, L and O
  • Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
  • The resulting tubes were labeled as P650 to P688.

Analytical digestion and gelelectrophoresis of P650-P688 with XbaI and PstI-HF

Investigator: Jeff, Rosario, Louise, Christopher

Aim of the experiment: Analytical digestion and gelelectrophoresis of P650-P688 with XbaI and PstI-HF

Procedure:

  • batch for digestion of P650-P688
volume reagent
5 µl Plasmid DNA
1 µl Cut Smart buffer
0.2 µl XbaI
0.2 µl PstI-HF
3.6 µl ddH2O
=10 µl TOTAL


  • Analytical digestion was performed at 37 °C for 1 h.
  • After incubation 2  µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.
  • Analytical gelelectrophoresis was performed at 90 V for 1 h.

Saturday, August 17th

Picking of of E. coli XL1 blue transformed with P449, P541, F230+F231, 2x K-Platte, 1x L-Platte, 2x M-Platte, 1x O-Platte

Investigator: Andi, Johanna

Aim of the experiment: Picking of of E. coli XL1 blue transformed with P449, P541, F230+F231, 2x K-Platte, 1x L-Platte, 2x M-Platte, 1x O-Platte

Procedure:

  • F230+F231: 8 Colonies were picked from chloramphenicol plates.
  • P449: 4 Colonies were picked from chloramphenicol plates.
  • P541: 4 Colonies were picked from ampicilin plates.
  • K-Plate: 4 Colonies were picked from chloramphenicol plates.
  • L-Plate: 4 Colonies were picked from chloramphenicol plates.
  • M-Plate: 4 Colonies were picked from chloramphenicol plates.
  • O-Plate: 4 Colonies were picked from chloramphenicol plates.
  • Each colony was transferred into a tube containing a relation of 1:1000 of LB-medium : antibiotic
  • These tubes were transferred into a cell culture shaker at 37 °C and were incubated 16 h

Preparative digestion of P625, P516, P644 + Preparative Gelelectrophoresis + Gelextraction

Investigator: Andi, Johanna

Aim of Experiment: Obtaining digested fragments of P625 (SERK-SigP_EreB_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P516 (SERK-SigP_FluA_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1)and P644 (Banana odour BBa_J45014)

Procedure:

Reaction batch for P625, P516 and P644:

volume reagent
20 µl PXXX
4 µl CutSmart Buffer (10x)
2 µl EcoRI (20 U/µl)
2 µl PstI (20 U/µl)
12 µl ddH2O
=40 µl TOTAL
  • Reaction batches were incubated at 37 °C for 2.5  h.

20130817 P516 P625 P644 EcoRI.PstI.png

  • Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.
  • Gelextraction was performed after preparative Gelelectrophoresis
  • The resulting fragments were named F234 (digestion of P516), F235 (digestion of P625), F236 (digestion of P644).

Ligation of F230+ F234, F230+ F235, F230+F236

Investigator: Johanna, Andi

Aim of the experiment: Ligation of F230+ F234, F230+ F235, F230+F236

Procedure:

Ligation batch for F230+F234, vector:insert = 1:3

volume reagent
6.3 µl F234(228 ng/µl, 1600 bp)
0.4 µl F230 (100 ng/µl,  bp)
10.3 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F230+F235, vector:insert = 1:3

volume reagent
9.0 µl F235(214 ng/µl, 2300 bp)
0.4 µl F230 (100 ng/µl,  bp)
7.6 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL

Ligation batch for F230+F236, vector:insert = 1:3

volume reagent
16.6 µl F236(330 ng/µl, 1500 bp)
0.4 µl F230 (100 ng/µl,  bp)
0.0 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • neg. ctrl. w/ water instead of insert
  • Ligation was performed at room temperature, 1 h

Miniprep of F230+F231 (version 1-4), P541 (version 1-2), K3, K4, L2, L3, M3, M4, O2, O3, P3-P10 and P449 (version 1-2)

Investigator: Johanna, Andi

Aim of the experiment: Miniprep of F230+F231 (version 1-4), P541 (version 1-2), K3, K4, L2, L3, M3, M4, O2, O3, P3-P10 and P449 (version 1-2)

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
  • The new plasmids were numbered as followed:
Plasmid Miniprep-Plasmid
F230+F231 (version 1) P689
F230+F231 (version 2) P690
F230+F231 (version 3) P691
F230+231 (version 4) P692
P541 (version 1) P693
P541 (version 2) P694
K3 P695
K4 P696
L2 P697
L3 P698
M3 P699
M4 P700
O2 P701
O3 P702
P3 P703
P4 P704
P5 P705
P6 P706
P7 P707
P8 P708
P9 P709
P10 P710
P449 (version 1) P711
P449 (version 2) P712

Analytical digestion and gelelectrophoresis of P689-P712 with XbaI and PstI

Investigator: Andi, Johanna

Aim of the experiment: Analytical digestion and gelelectrophoresis of P689-P712 with XbaI and PstI

Procedure:

  • batch for digestion of P689-712
volume reagent
5 µl Plasmid DNA
1 µl Cut Smart buffer
0.2 µl XbaI-HF
0.2 µl PstI-HF
3.6 µl ddH2O
=10 µl TOTAL
  • Analytical digestion was performed at 37 °C for 1.5 h.
  • After incubation 2  µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.
  • Analytical gelelectrophoresis was performed at 90 V for 1 h.

TUM13 20130818 anal verdau P689 bis P698 XbaI PstI.png

Lane 1kB DNA ladder Digestion of P689 Digestion of P690 Digestion of P691 Digestion of P692 Digestion of P693 Digestion of P694 Digestion of P695 Digestion of P696 Digestion of P697 Digestion of P698
Results as expected as expected as expected as expected as expected weird weird as expected as expected as expected weird

TUM13 20130818 anal verdau P699 bis P708 XbaI PstI.png

Lane 1kB DNA ladder Digestion of P699 Digestion of P700 Digestion of P701 Digestion of P702 Digestion of P703 Digestion of P704 Digestion of P705 Digestion of P706 Digestion of P707 Digestion of P708
Results as expected as expected as expected weird as expected as expected as expected as expected as expected as expected as expected

20130818 anal verdau P709 bis P712 XbaI PstI.jpg

Lane 1kB DNA ladder Digestion of P709 Digestion of P710 Digestion of P711 Digestion of P712
Results as expected as expected as expected as expected as expected

Transformation of E. coli XL1-Blue with ligation product F230+F234, F230+F235, F230+F236

Investigator: Johanna, Andi

Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product F230+F234, F230+F235, F230+F236.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 1,5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Sunday, August 18th

BITTE VERVOLLSTÄNDIGEN (P-Nummern) Midiprep of Safety_PIF3, PP1 membrane bound, Safety_PIF6, Safety_PIF3, Positve control mit PppActin5, EreB cyt., NanoLuc cyt., XylE cyt., NanoLuc sek. (IgKappa), NanoLuc sek. (SERK), GFP cyt., GFP sek. (IgKappa), FluA membrane bound, stress-inducible promoter, GST, NanoLuc membrane bound

Investigator: Andreas, Rosario

Aim of the experiment: Midiprep of RFC25 compatible RFP generator in (pSB1C3)

Procedure:

  • Midiprep was performed after manufacturer's protocol (QIAprep Midiprep, QIAGEN)

Digestion of BITTE VERVOLLSTÄNDIGEN

Investigator: Ingmar

Aim of the experiment:

Procedure:

Week 18

Monday, August 19th

Analytical Gelelectrophoresis of digested Midi-Preps F243 - F260

Investigator: Ingmar

Aim of the experiment: Verification of linearisation of digest fragments F234-F260

Procedure:

  • Concentrations of digestions were measured (blanked against EB-buffer), see Inventory List
  • 5 µl of DNA loading buffer (10x) mixed with 1 µl Midiprep-DNA, on 1% agarose gel.
  • 90 V 1 h.

20130819 F234-F244.png

Lane 1kB DNA ladder F234 F235 F236 F237 F238 F239 F240 F241 F242 F243 F244
Name - Nanoluc secretory GFP cytoplasmatic Nanoluc membrane bound Nanoluc cytoplasmatic Ere B cytoplasm PIF6 PIF3 clone1 PIF3 clone2 GFP_IgKappa PP1 C1
Results

20130819 F245-F252.png

Lane 1kB DNA ladder F245 F246 F247 F248 F249 F250 F251 F252
Name - Safety_PIF3 PP1 mebrane bound Safety_PIF6 Safety_PIF? pos ctrl mit Ppp Actin5 Ere B cytoplasm Nanoluc cytoplasm XylE cytoplasm
Results

20130819 F253-F260.png

Lane 1kB DNA ladder F253 F254 F255 F256 F257 F258 F259 F260
Name - Nanoluc secretory IgKappa Nanoluc secretory SERK GFP cytoplasm GFP secretory IgKappa FluA membrane bound stress inducible promotor GST Nanoluc membrane bound
Results -

Preparation of the linearized DNA (F234-F260) for the Moss Transformation

Investigator: Ingmar

Aim of the experiment: Preparation of the linearized DNA (F234-F260) for the Moss Transfection

Procedure:

  • The DNA pellets were solved in 500 µl ddH2O
  • Linerization of the DNA with EcoRI: 56,6 µl CutSmart buffer and 10 µl EcoRI were added to the 500 µl DNA. Incubation over night at 37 °C.
  • For purification the DNA was again precipitated with pure isopropanol: Addition of 500 µl isopropanol, mixing by inverting, centrifugation at 16,200 g for 40 min; supernatant was discarded
  • Wash pellet with 200 µl 70 % ethanol, centrifugation at 16,200 g for 40 min, supernatant discarded. The pellet was air dried at 50 °C for 8 min.
  • The DNA pellets were dissolved in the respectively calculated volumes of 0,1 M Ca(NO3)2 soultion to obtain final concentrations of 250 ng/µl.
fragment name concentration concentration w/ loss volume 0,1 M Ca(NO3)2
F234 Nanoluc SERK-SigP 440,8 396,72 872,78
F235 GFP cytoplasm 241,8 217,62 478,76
F236 Nanoluc membrane 339,9 305,91 673,00
F237 Nanoluc cytoplasm 219,1 197,19 433,82
F238 EreB cytoplasm 355,9 320,31 704,68
F239 PIF6 669,9 602,91 1326,40
F240 PIF3 (1) 438,1 394,29 867,438
F241 PIF3 (2) 642,9 578,61 1272,942
F242 GFP IgKappa 339,4 305,46 672,012
F243 PP1 receptor 333,5 300,15 660,33
F244 Catecholdioxygenase cytoplasm 342,4 308,16 677,952

Tuesday, August 20th

Preparative digestion + Gelelectrophoreses + Gelextraction of P643/F261

Investigator: Andi, Rosario, Johanna

Aim of the experiment: Preparative digestion + Gelelectrophoreses + Gelextraction of P643/F261.

Procedure:

Reaction batch for P643:

volume reagent
20 µl P643
5 µl CutSmart Buffer (10x)
2 µl EcoRI (20 U/µl)
2 µl NgoMIV (20 U/µl)
12 µl ddH2O
=40 µl TOTAL

Result of the Gelelectrophoresis:

TUM13 20130820 prepgel P643 EcoRI.NgoMIV.png

  • Reaction batch was incubated at 37 °C for 2 h.
  • Digested product was purified with QIAquick PCR Purification Kit, QIAGEN.
  • Gelelectrophoresis was performed for 1 h and 90V.
  • Gelextraction was performed after manufacturers protocol and the resulting Fragment was called F261.

Sequencing of P643 (SYFP2+ G-TEV-site-G)

Investigator: Andi

Aim of the experiment: Sequencing of P643 (SYFP2+ G-TEV-site-G)

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label Barcode Primer
P643 FR0223317 03

Ligation of F261+F225 (G-TEV-site-G_SYFP2 + eCFP)

Investigator: Rosario, Andi, Johanna

Aim of the experiment: Ligation of F261+F225 (G-TEV-site-G_SYFP2 + eCFP)

Procedure:

Ligation batch for F261+F225, vector:insert = 1:3

volume reagent
1,5 µl F225
1,2 µl F261
14,3 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • neg. ctrl. w/ water instead of insert
  • Ligation was performed at room temperature, 1 h

Transformation of E. coli XL1-Blue with ligation product F225+F261 and Retrafo of P643

Investigator: Rosario

Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product F225+F261 and Retrafo of P643.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 5 µl of DNA (Ligation) and 1 µl of DNA (Retrafo) were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Transformation of Physcomitrella patens with F234-F260

Investigator: Ingmar, Jeff

Aim of the experiment: Transformation of Physcomitrella patens with F234-F260

Procedure:

According to the Reski protocol [http://www.plant-biotech.net/]

  • Prepare 4&nbsp% driselase solution in 0.5&nbspM mannitol, vortex, keep on rotating platform for 45&nbspmin (protected from light) and centrifuge at 3500 rpm, 10 min; sterile filter
  • Extraction of moss from bioreactor culture by filtering through protoplast sieve (mesh size 100 µm)
  • Addition of 12 ml 0.5 M mannitol solution and 4 ml of driselase stock solution (final concentration: 1 % w/v) and Incubation for 2 h at room temperature on rotating platform (protected from light)
  • Gently filter moss material through protoplast sieve (mesh size 100 µm), then filtrate through 50 µm sieve
  • Centrifugation at 500 rpm for 10 min with slow acceleration rates in a glass tube and discard supernatant; wash protoplasts with mannitol solution, repeat.
  • Resuspend pellet in total 10 ml mannitol
  • Determine concentration of protoplasts/ ml
  • 100&nbspµl of 250 ng/µl DNA solution with 350 µl PEG 4000 solution (8 g PEG4000 ad 20 g 3M medium, sterile filtered), incubate 30 min while gently mixing every 5 min
  • Dilution with 3M medium every 5 min: 1 ml, then 2 ml, then 3, then 4
  • Centrifugation at 500 rpm for 10 min, discard supernatant, resuspend in 4 ml regeneration medium and divide into 2 6wells
  • Incubation in the dark over night, then light/dark regime of 16/8 h

Wednesday, August 21st

Picking of of E. coli XL1 blue transformed with ligation product F225+F261 and Retrafo of P643

Investigator: Rosario

Aim of the experiment: Picking of of E. coli XL1 blue transformed with ligation product F225+F261 and Retrafo of P643.

Procedure:

  • F225+F261: 3 Colonies were picked from chloramphenicol plates.
  • P643: 1 Colony was picked from a chloramphenicol plate.
  • Each colony was transferred into a tube containing a relation of 1000:1 of LB-medium : antibiotic
  • These tubes were transferred into a cell culture shaker at 37 °C and were incubated 12 h


Transformation of E. coli XL1-Blue with ligation product F225+F261

Investigator: Rosario

Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product F225+F261.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Thursday, August 22nd

Miniprep of E. coli Xl1-Blue transformed with ligation product F261 + F225 and Retrafo of P643

Investigator: Florian

Aim of the experiment: Miniprep of E. coli Xl1-Blue transformed with ligation product F261 + F225 and Retrafo of P643.

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
  • The resulting tubes were labeled as P714 to P716.

Analytical digestion and gelelectrophoresis of P714 & P715

Investigator: Florian

Aim of the experiment: Analytical digestion and gelelectrophoresis of P714 & P715.

Procedure:

Batch for digestion with EcoRI & PstI

volume reagent
2.5 µl Plasmid
2 µl Cut Smart buffer
0.25 µl EcoRI-HF
0.25 µl PstI-HF
15 µl ddH2O
=20 µl TOTAL
  • Analytical digestion was performed at 37 °C for 1 h.
  • After incubation 2  µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.
  • Analytical gelelectrophoresis was performed at 90 V for 1 h.

TUM13 20130822 anal Verd P714-P715 EcoRI.PstI.png

Lane: 2log DNA ladder P714 P715
Result: as expected as expected


ReTrafo of E. coli XL1-Blue with P650, P652,P654, P656, P658, P661, P663, P666, P672, P678, P679, P682, P685, P706, P709, P625, P414, P628, P629, P630, P632, P713

Investigator: Rosario

Aim of the experiment: ReTrafo of E. coli XL1-Blue with P650, P652,P654, P656, P658, P661, P663, P666, P672, P678, P679, P682, P685, P706, P709, P625, P414, P628, P629, P630, P632, P713.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 5 µl of DNA (Ligation) and 1 µl of DNA (Retrafo) were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Preparative digestion of P625, P414 with EcoRI, PstI and MlyI, P628, P629, P630, P632 with EcoRI, PstI and SspI + Preparative Gelelectrophoresis + Gelextraction

Investigator: Johanna, Louise


Procedure:

Reaction batch for P625, P414:

volume reagent
20 µl Plasmid DNA
4 µl CutSmart Buffer (10x)
1 µl EcoRI (20 U/µl)
1 µl PstI (20 U/µl)
2 µl MlyI (20 U/µl)
12 µl ddH2O
=40 µl TOTAL

Reaction batch for P628,P629,P630, P632:

volume reagent
20 µl Plasmid DNA
4 µl CutSmart Buffer (10x)
1 µl EcoRI (20 U/µl)
1 µl PstI (20 U/µl)
2 µl SspI (20 U/µl)
12 µl ddH2O
=40 µl TOTAL
  • Reaction batches were incubated at 37 °C for 2.5  h.
  • Preparative gelelectrophoresis was performed at 90 V for 1 h in an 0.5% agarose gel.

TUM13 20130822 prepgel P625 EcoRI.PstI.MlyI P628 P629 EcoRI.PstI.SspI.png

TUM13 20130822 prepgel P630 EcoRI.PstI.SspI P632 EcoRI.PstI.SspI P414 EcoRI.PstI.MlyI.png

  • Gelextraction was performed after preparative Gelelectrophoresis
  • The resulting fragments were named F262 (digestion of P625), F264 (digestion of P628), F263 (digestion of P629, F265 (digestion of P630), F266 (digestion of P632) and F267 (digestion of P414).

Ligation of F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267

Investigator: Flo, Rosario, Jeff

Aim of the experiment: Ligation of F230+F262 (SERK-SigP_EreB_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3), F230+F263 (SERK-SigP_SpyTag_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_NanoLuc_SpyCatcher in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3), F230+F264 (SERK-SigP_SpyCatcher_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_SpyTag_NanoLuc in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3), F230+F265 (SERK-SigP_SpyCatcher_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_NanoLuc_SpyTag in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3), F230+F266 (SERK-SigP_SpyTag_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_SpyCatcher_NanoLuc in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3), F230+F267 (SERK-SigP_Laccase_Strep-tag-II-TEV-site-Linker_SERK-TMD_8AALinker_GFPmut1 in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3)

Procedure:

  • Ligation batch for F230+F262, vector:insert = 1:3
volume reagent
0.4 µl F230
15.96 µl F268
0.7 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F230+F263
volume reagent
0.4 µl F230
16.6 µl F263
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F230+F264, vector:insert = 1:3
volume reagent
0.4 µl F230
5.9 µl F264
10.7 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F230+F265, vector:insert = 1:3
volume reagent
0.4 µl F230
3.6 µl F265
13 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F230+F266, vector:insert = 1:3
volume reagent
0.4 µl F230
2.5 µl F266
14.1 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F230+F267, vector:insert = 1:3
volume reagent
0.4 µl F230
9.8 µl F267
6.8 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • neg. ctrl. water instead of insert.
  • Ligation was performed at room temperature for 1 h.

Friday, August 23rd

Preparative digestion of P715 with XbaI and PstI + Preparative Gelelectrophoresis + Gelextraction

Investigator: Louise, Flo


Procedure:

Reaction batch for P715:

volume reagent
20 µl Plasmid DNA
4 µl CutSmart Buffer (10x)
1 µl XbaI (20 U/µl)
1 µl PstI (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Reaction batches were incubated at 37 °C for 2.5  h.
  • Preparative gelelectrophoresis was performed at 90 V for 1 h in an 0,5% agarose gel.

TUM13 20130823 prep Verd P715 XbaI.PstI.png

  • The lower band was cut out
  • Gelextraction was performed after preparative Gelelectrophoresis

Transformation of E. coli XL1-Blue with P6 (pGAL in pTUM 100)

Investigator: Louise

Aim of the experiment: Transformation of E. coli XL1-Blue with P6 (pGAL in pTUM 100). Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new ampicillin plates.

Sequencing of P715 (sYFP_G_TEV-site_G_cYFP)

Investigator: Louise

Aim of the experiment: Sequencing of P715 (sYFP_G_TEV-site_G_cYFP)

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The gene we sequenced received the following barcode:

Label Barcode Primer
P715 FR02233118 03

Ligation of F187+F268 and F188+F268

Investigator: Flo

Aim of the experiment: Ligation of F187+F268 and F188+F268

Procedure:

  • Ligation batch for F187+F268, vector:insert = 1:3
volume reagent
0.527 µl F187
1.406 µl F268
15.067 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F188+F268, vector:insert = 1:3
volume reagent
0.732 µl F188
1.406 µl F268
14.862 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL


  • neg. ctrl. water instead of insert
  • Ligation was performed at room temperature for 1 h


Picking of transformed Plasmid E. coli XL1 blue with P650, P652,P654, P656, P658, P661, P663, P666, P672, P678, P679, P682, P685, P706, P709, P625, P414, P628, P629, P630, P632, P713

Investigator: Louise

Aim of the experiment: Picking of transformed Plasmid E. coli XL1 blue with P650, P652,P654, P656, P658, P661, P663, P666, P672, P678, P679, P682, P685, P706, P709, P625, P414, P628, P629, P630, P632, P713

Procedure:

  • Picking and overnight culture after standard laboratory's protocol. (6 µl Cam, 6 ml LB-medium)
  • 1 colony was picked for every transformation product.

Transformation of E. coli XL1-Blue with ligation product F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267

Investigator: Rosario

Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Inoculation of 50 ml LB meida wirh E. coli XL1 blue transformed with P650, P652,P654, P656, P658, P661, P663, P666, P672, P678, P679, P682, P685, P706, P709, P625, P414, P628, P629, P630, P632, P713

Investigator: Ingmar

Aim of the experiment: Inoculation of Midiprepcultures of E. coli XL1 blue transformed with P650, P652,P654, P656, P658, P661, P663, P666, P672, P678, P679, P682, P685, P706, P709, P625, P414, P628, P629, P630, P632, P713

Procedure:

  • 1 ml of each of the 6 ml LB Miniprepcultures prepared by Louise was used to inoculate 50 ml Lb media with 50 µl Cam. Incubation overnight in a Minitron shaker at 37 °C and 160 RPM


Saturday, August 24th

Miniprep of E. coli XL1-Blue transformed with ligation products P650, P652,P654, P656, P658, P661, P663, P666, P672, P678, P679, P682, P685, P706, P709, P625, P414, P628, P629, P630, P632, P713

Investigator: Jeff, Rosario

Aim of the experiment: Miniprep of E. coli XL1-Blue transformed with ligation products P650, P652,P654, P656, P658, P661, P663, P666, P672, P678, P679, P682, P685, P706, P709, P625, P414, P628, P629, P630, P632, P713

Procedure:

  • Ligation products were labelled B to V (in the same order as in Aim of the experiment, above)
  • Two clones for every ligation product except for H, L and O
  • Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
  • The resulting tubes were labeled as P650 to P688.

Preparation of media for competent cells

Investigator : Johanna, Louise

Aim of the experiment:Preparation of media for competent cells

Procedure:

  • Picking of one colony of overnight stem culture of non competent XL1-Blue E.Coli in 50 ml LB-Medium without antibiotics (under the sterile bench)
  • this was incubate overnight at 37 °C
  • Batch for 1l of a 0.1 M MgCl2-solution
amount reagent
20.33 g MgCl2 (M=203.3 g/mol)
1 l ELGA H2O
  • Batch for 500ml of a 50 mM CaCl2-solution
amount reagent
3.67 g CaCl2 (M=147.02 g/mol)
500 ml ELGA H2O
  • Batch for 50ml of a 50 mM CaCl2-solution, 15% v/v Glycerin
amount reagent
0.368 g CaCl2 (M=147.02 g/mol)
0.458 g Glycerin (density=1.26)
50 ml ELGA H2O
  • Autoclave each bottle of solution and leave each in a 4°C room.


Transformation of E. coli XL1-Blue with P496, P661, P629

Investigator: Rosario

Aim of the experiment: Transformation of E. coli XL1-Blue with P496, P661, P629.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Picking of transformed Plasmid E. coli XL1 blue with ligation product F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267, F187+F268 and F188+F268

Investigator: Rosario

Aim of the experiment: Picking of transformed Plasmid E. coli XL1 blue with ligation product F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267, F187+F268 and F188+F268

Procedure:

  • Picking and overnight culture after standard laboratory's protocol. (4 µl Cam, 4 ml LB-medium)
  • 3 colones were picked for every transformation product, 2 for F188+F268 and F187+F268.

Ligation of F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267

Investigator: Jeff

Aim of the experiment: Ligation of F230+F262 (SERK-SigP_EreB_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3), F230+F263 (SERK-SigP_SpyTag_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_NanoLuc_SpyCatcher in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3), F230+F264 (SERK-SigP_SpyCatcher_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_SpyTag_NanoLuc in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3), F230+F265 (SERK-SigP_SpyCatcher_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_NanoLuc_SpyTag in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3), F230+F266 (SERK-SigP_SpyTag_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_SpyCatcher_NanoLuc in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3), F230+F267 (SERK-SigP_Laccase_Strep-tag-II-TEV-site-Linker_SERK-TMD_8AALinker_GFPmut1 in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3)

Procedure:

  • Ligation batch for F230+F262, vector:insert = 1:3
volume reagent
0.4 µl F230
15.96 µl F268
0.7 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F230+F263
volume reagent
0.4 µl F230
16.6 µl F263
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F230+F264, vector:insert = 1:3
volume reagent
0.4 µl F230
5.9 µl F264
10.7 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F230+F265, vector:insert = 1:3
volume reagent
0.4 µl F230
3.6 µl F265
13 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F230+F266, vector:insert = 1:3
volume reagent
0.4 µl F230
2.5 µl F266
14.1 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F230+F267, vector:insert = 1:3
volume reagent
0.4 µl F230
9.8 µl F267
6.8 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • neg. ctrl. water instead of insert.
  • Ligation was performed at 16 °C overnight.

Midiprep and preparative digestion of cultures with E. coli XL1 blue transformed transformed with P650, P652,P654, P656, P658, P661, P663, P666, P672, P678, P679, P682, P685, P706, P709, P625, P414, P628, P629, P630, P632, P713

Investigator: Ingmar & Louise

Aim of the experiment: Production of DNA for moss transformations with P650, P652,P654, P656, P658, P661, P663, P666, P672, P678, P679, P682, P685, P706, P709, P625, P414, P628, P629, P630, P632, P713

Procedure:

  • The preparation of plasmid DNA was executed according to the user manual provided by Qiagen. The final DNA pellet was dissolved in 500 µl EB buffer.
  • The plasmid DNA was linearized with EcoRI HF at 37 °C overnight in batches with the following composition:
Component Volume [µl]
EcoRI HF5
DNA500
CutSmart56


Sunday, August 25th

Preparation of competent cells according to the CaCl 2-method (Cohen et al.,1972)

Investigator: Johanna, Christopher, Louise


Aim of the experiment: Preparation of competent cells according to the CaCl 2-method (Cohen et al.,1972)

Procedure:

  • Add Tetracyclin (ratio: 1:1000) to the 2YT-medium
  • Measure the OD550 of it until OD550=0.5
  • Put the culture in Falcon tubes (leaving on ice)
  • Centrifuge the falcon tubes by 5000 rpm, 4°C, 10 min
  • Resuspend the falcon tubes each with 40 ml 0,1 M MgCl2 solution after removing the supernatants
  • Centrifuge the falcon tubes by 5000 rpm, 4°C, 10 min
  • Resuspend the falcon tubes each with 20 ml 50mM CaCl2 solution after removing the supernatants
  • Incubate the falcon tubes on ice for 30 min
  • Centrifuge the falcon tubes by 5000 rpm, 4°C, 10 min
  • Resuspend the falcon tubes each with 2 ml 50mM CaCl2, 15 % v/v Glycerin solution after removing the supernatants. From this step everything needs to be carry out in a 4°C room.
  • Mix the solutions of each falcon tube and transfer the content in eppis (each with 100 and accordingly 150 µl)
  • Store the boxes of eppis at -80 °C

Ligation of F230+F228

Investigator: Johanna

Aim of the experiment: Ligation of F230+F228

Procedure:

  • Ligation batch for F230+F228, vector:insert = 1:3
volume reagent
0.41 µl F230(243.7 ng/µl; 5796 bp)
3.21 µl F228(25.5 ng/µl; 1581 bp)
13.38 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • neg. ctrl. water instead of insert.
  • Ligation was performed at room temperature for 1 h.

Analytical gelelectrophoresis of P742-P757,P759, P760

Investigator: Johanna

Aim of the experiment: Analytical gelelectrophoresis of P742-P757,P759, P760

Procedure:

  • Procedure of a mixture: 300 µl H2O + 50 µl DNA loading buffer (10x)
  • 8  µl of DNA loading buffer (10x) were added to 1 µl Plasmid and were loaded on a 1% agarose gel.
  • Analytical gelelectrophoresis was performed at 90 V for 1 h.

20130825 analgel P748-P753 EcoRI.PstI.png

Lane: 2log DNA ladder P748 P749 P750 P751 P752 P753
Result: as expected as expected as expected as expected as expected as expected

20130825 analgel P754-P757 P759 P760 EcoRI.png

Lane: 2log DNA ladder P754 P755 P756 P757 P759 P760
Result: as expected as expected as not expected as not expected as not expected as not expected

20130825 analgel P742-P747EcoRI.PstI.png

Lane: 2log DNA ladder P742 P743 P744 P745 P746 P747
Result: as expected as expected as expected as expected as expected as expected

Transformation of E. coli XL1-Blue with ligation product F230+F228

Investigator: Johanna

Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product F230+F228.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Miniprep of ligations F187+F268 (PIF3 + fret system) and F188+F268 (PIF6 + fret system) and analytical digest with EcoRI & PstI

Investigator: Jeff

Aim of the experiment: Obtaining ligated plasmids F187+F268 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES + sYFP_G_TEV-site_G_cYFP) (= P761 and P762) and F188+F268 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES + sYFP_G_TEV-site_G_cYFP) (= P763 and P764)

Procedure:

  • Preparation of the plasmids according to the QIAGEN Miniprep Kit, elution in 50 µl

Batch for digestion with EcoRI & PstI

volume reagent
2.5 µl Plasmid
2 µl Cut Smart buffer
0.25 µl EcoRI-HF
0.25 µl PstI-HF
15 µl ddH2O
=20 µl TOTAL
  • Analytical digestion was performed at 37 °C for 1 h.
  • 1 % agarose gel with ethidium bromide, 90 V, 30 min
  • Ctrl: F187 and F188 respectively

TUM13 20130825 analVerd P761 P762 EcoRI.PstI F187 P763 P764 EcoRI.PstI F188.png

Lane: 2log DNA ladder P761 dig. EcoRI&PstI P762 dig. EcoRI&PstI F187 P763 dig. EcoRI&PstI P764 dig. EcoRI&PstI F188
Result: - not as expected not as expected ctrl not as expected not as expected ctrl

Preparative digestion of P761-P764 with EcoRI and PstI + preparative gelelectrophoresis + gelextraction

Investigator: Jeff

Aim of the experiment: Obtaining fragments of PIF3/PIF6 with fret system for ligation into expression vector

Procedure:

Reaction batch for P761-P764:

volume reagent
20 µl Plasmid DNA
4 µl CutSmart Buffer (10x)
1 µl EcoRI (20 U/µl)
1 µl PstI (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Reaction batches were incubated at 37 °C for 2.5  h.
  • Preparative gelelectrophoresis was performed at 90 V for 1 h in an 0,5% agarose gel.
  • Analytical digest shows failed ligaton, preparative digest was cancelled

Transformation of E. coli XL1-Blue new competent cells with P649 (Retrafo)

Investigator: Louise

Aim of the experiment: Test-Transformation with new batch of competent cells

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.
  • The negative control of competent cells which were not transfomed with Plasmid DNA but with water, were plated on all kind of plates: Chloramphenicol, Kanamycin, Ampicillin and tetracyclin

Troubleshooting: Repeat of ligation of F187+F268 and F188+F268 and re-picking of the 08/23 ligation

Investigator: Jeff

Aim of the experiment: Ligation of F187+F268 and F188+F268

Procedure:

  • Ligation batch for F187+F268, vector:insert = 1:3
volume reagent
0.53 µl F187
1.41 µl F268
15.067 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F188+F268, vector:insert = 1:3
volume reagent
0.73 µl F188
1.41 µl F268
14.862 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • neg. ctrl. water instead of insert
  • Ligation was performed at 16 °C over night

Picking of transformed Plasmid E. coli XL1 blue with Retrafo of P661 (24.8.), P661 (22.8.), P629 and P496

Investigator: Florian

Aim of the experiment: Picking of transformed Plasmid E. coli XL1 blue with Retrafo of P661 (24.8.), P661 (22.8.), P629 and P496.

Procedure:

  • Picking and overnight culture after standard laboratory's protocol. (6 µl Cam, 6 ml LB-medium)
  • 2 colonies were picked for every transformation product.


Alcohol precipitation and purification of linearized Mididpreps of P650, P652,P654, P656, P658, P661, P663, P666, P672, P678, P679, P682, P685, P706, P709, P625, P414, P628, P629, P630, P632, P713

Investigator: Ingmar & Louise

Aim of the experiment: Purification of linearized DNA

Procedure:

  • 350 µl of 1M Sodiumactetat solution (pH adjusted to 5,2 with concentrated acetic acid) were added to each digestion sample.
  • The DNA was precipitated with 595 µl isopropanol analytical grade. Afterwards centrifugation at 15 000 xg for 30 min at 4°C.
  • After having removed the supernatant the DNA pellet was washed with 1 ml 70 % EtOH. Afterwards centrifugation at 15 000 xg for 30 min at 4°C.
  • The supernatant was removed, the samples once again centrifuged at 16 200 xg for 30 sec. and the accumulated supernatant again removed.
  • The pellets were air-dryed for 2 min under the maniar flow hood.
  • The DNA was solved in 100 µl sterile ddH2O and stored at -20 °C.


Analytical gelelectrophoresis of F228

Investigator: Florian

Aim of the experiment: Analytical gelelectrophoresis of F228.

Procedure:

  • 1  µl of DNA loading buffer (10x) were added to 4 µl Fragment and were loaded on a 1% agarose gel.
  • Analytical gelelectrophoresis was performed at 90 V for 1 h.

TUM13 20130825 anal Gel F228.png

Lane: 2log DNA ladder F228
Result: as expected

Transformation of E. coli XL1-Blue with ligation product F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267 and P711, P712, P713

Investigator: Jeff

Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267 and P711, P712, P713.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol (for P713 kanamycin) plates.


Week 19

Monday, August 26th

Miniprep of E. coli XL1-Blue transformed with Retrafo of P661 (24.8.), P661 (22.8.), P629 and P496

Investigator: Florian

Aim of the experiment: Miniprep of E. coli XL1-Blue transformed with Retrafo of P661 (24.8.), P661 (22.8.), P629 and P496.

Procedure:

  • Two clones for every ReTrafo
  • Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
  • The resulting tubes were labeled as P765 to P772.

Analytical digestion and gelelectrophoresis of P769-P776 with XbaI and PstI

Investigator: Johanna

Aim of the experiment: Analytical digestion and gelelectrophoresis of P769-P776 with XbaI and PstI

Procedure:

  • batch for digestion of P769-P776
volume reagent
2.5 µl Plasmid DNA
2 µl Cut Smart buffer
0.25 µl XbaI-HF
0.25 µl PstI-HF
15.0 µl ddH2O
=20 µl TOTAL
  • Analytical digestion was performed at 37 °C for 1 h.
  • After incubation 2  µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 0.5% agarose gel.
  • Analytical gelelectrophoresis was performed at 90 V for 1 h.

TUM13 20130826 analgel P769-P776 XbaI.PstI.png

Lane: 2log DNA ladder P769 P770 P771 P772 P773 P774 P775 P776
Result: as expected as expected as expected as expected as expected as expected as expected as expected

Miniprep of E. coli XL1-Blue transformed with F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267 and P711, P712, P713

Investigator: Florian

Aim of the experiment: Miniprep of E. coli XL1-Blue transformed with F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267 and P711, P712, P713.

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
  • The resulting tubes were labeled as P780 to P799.

Analytical digestion and gelelectrophoresis of P780 – P797

Investigator: Jeff

Aim of the experiment: Analytical digestion and gelelectrophoresis of P780 – P797.

Procedure:

Mastermix for analytical digestion with EcoRI and PstI

volume reagent
40 µl NEBuffer 3.1
5 µl XbaI
5 µl PstI
300 µl ddH2O
=350 µl TOTAL
  • 17.5 µl of the mastermix were mixedd together with the respective plasmids (P780 – P797).
  • Analytical digestion was performed at 37 °C for 1.5 h.
  • After incubation 2  µl of DNA loading buffer (10x) were added to the reaction batches and 10 µl were loaded on a 0.5% agarose gel.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.

TUM13 20130826 analgel P780-P790 XbaI.PstI.png

Lane: 2 log DNA ladder P780 digested with XbaI & PstI P781 digested with XbaI & PstI P782 digested with XbaI & PstI P783 digested with XbaI & PstI P784 digested with XbaI & PstI P785 digested with XbaI & PstI P786 digested with XbaI & PstI P787 digested with XbaI & PstI P788 digested with XbaI & PstI P789 digested with XbaI & PstI P790 digested with XbaI & PstI
Result: as expected corrupt corrupt corrupt as expected as expected as expected as expected as expected corrupt corrupt

TUM13 20130826 analgel P791-P797 XbaI.PstI.png

Lane: 2 log DNA ladder P791 digested with XbaI & PstI P792 digested with XbaI & PstI P793 digested with XbaI & PstI P794 digested with XbaI & PstI P795 digested with XbaI & PstI P796 digested with XbaI & PstI P797 digested with XbaI & PstI
Result: corrupt corrupt corrupt as expected as expected as expected as expected


Picking of transformed Plasmid E. coli XL1 blue with ligation product F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267, F187+F268, F188+F268, F230+F228 and Retrafos of P711, P712, P713, P649

Investigator: Rosario

Aim of the experiment: Picking of transformed Plasmid E. coli XL1 blue with ligation product F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267, F187+F268,F188+F268, F230+F228 and Retrafos of P711, P712, P713, P649

Procedure:

  • Picking and overnight culture after standard laboratory's protocol. (4 µl Cam, 4 ml LB-medium)
  • 3 colones were picked for every ligation product (5 for F187+F268,F188+F268) , 2 for the Retrafos.

Tuesday, August 27th

Transformation of E. coli XL1-Blue with ligation products F187+F268 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES_eCFP_G_TEV-site_G_SYFP2) and F188+F268 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES_eCFP_G_TEV-site_G_SYFP2)

Investigator: Jeff

Aim of the experiment: Transformation of E. coli XL1-Blue with ligation products F187+F268 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES_eCFP_G_TEV-site_G_SYFP2) and F188+F268 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES_eCFP_G_TEV-site_G_SYFP2).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 90 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Picking of transformed Plasmid E. coli XL1 blue with P6,P773-P776

Investigator: Johanna

Aim of the experiment: Picking of transformed Plasmid E. coli XL1 blue with P6,P773-P776

Procedure:

  • P773-P776: 2 Colonies were picked from chloramphenicol plates.
  • P6: 2 Colonies were picked from ampicilin plates.
  • P773-P776: Each colony was transferred into a tube containing 6 mL of LB-medium + 6 µL chloramphenicol(1000x).
  • P6: Each colony was transferred into a tube containing 6 mL of LB-medium + 6 µL ampicilin(1000x).
  • These tubes were transferred into a cell culture shaker at 37 °C and were incubated 12 h

Miniprep of E. coli XL1-Blue transformed with P649, F230+F196, F230+262, F230+263, F230+264, F230+266, F230+267

Investigator: Rosario

Aim of the experiment: Miniprep of E. coli XL1-Blue transformed with P649, F230+F196, F230+262, F230+263, F230+264, F230+266, F230+267.

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
  • The resulting tubes were labeled as P800 to P809.

Transformation of E. coli XL1-Blue with P794

Investigator: Rosario

Aim of the experiment: Transformation of E. coli XL1-Blue with P794.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Preparative digestion of P290, P387, P787 with EcoRI and PstI, and preparative digestion of P799 with XbaI and PstI + Preparative Gelelectrophoresis + Gelextraction

Investigator: Rosario

Procedure:

Reaction batch for P290/P387/P787:

volume reagent
20 µl Plasmid DNA
4 µl NEB 2.1 Buffer (10x)
1 µl EcoRI-HF (20 U/µl)
1 µl PstI (20 U/µl)
14 µl ddH2O
=40 µl TOTAL

Reaction batch for P799:

volume reagent
20 µl Plasmid DNA
4 µl NEB 3.1 Buffer (10x)
1 µl XbaI (20 U/µl)
1 µl PstI (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Reaction batches were incubated at 37 °C for 3  h.
  • Preparative gelelectrophoresis was performed at 90 V for 1 h in an 0,5% (P787) and 1% agarose gel.

TUM13 20130827 prepgel P290,P787,P387 EcoRI.PstI.png

  • The results were corrupt so the experiment was repeated

TUM13 20130827 prepgel P799 XbaI.PstI.png


  • The results were corrupt so the experiment was repeated

Analytical digestion and gelelectrophoresis of P800 – P809

Investigator: Rosario

Aim of the experiment: Analytical digestion and gelelectrophoresis of P800 – P809.

Procedure:

Mastermix for analytical digestion with EcoRI and PstI

volume reagent
24 µl NEBuffer 3.1
3 µl XbaI
3 µl PstI
180 µl ddH2O
=210 µl TOTAL
  • 17.5 µl of the mastermix were mixedd together with the respective plasmids (P780 – P797).
  • Analytical digestion was performed at 37 °C for 1.5 h.
  • After incubation 2  µl of DNA loading buffer (10x) were added to the reaction batches and 10 µl were loaded on a 1% agarose gel.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.

TUM13 20130827 analgel P800-P809 XbaI.PstI.png

Lane: 2 log DNA ladder P800 digested with XbaI & PstI P801 digested with XbaI & PstI P802 digested with XbaI & PstI P803 digested with XbaI & PstI P804 digested with XbaI & PstI P805 digested with XbaI & PstI P806 digested with XbaI & PstI P807 digested with XbaI & PstI P808 digested with XbaI & PstI P809 digested with XbaI & PstI
Result: as expected corrupt corrupt corrupt as expected as expected as expected as expected as expected corrupt


Retransformation of E. coli XL1-Blue with P290, P387, P787, P799

Investigator: Rosario

Aim of the experiment: Retransformation of E. coli XL1-Blue with P290, P387, P787, P799.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 90 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates (Kanamycin for P799).

Picking of Ligation F228+F230 (banana cytoplasm. + plasmid w/ Mini MCS)

Investigator: Rosario

Aim of the experiment:

  • Picking of Ligation F228+F230 (banana cytoplasm. + plasmid w/ Mini MCS)


Procedure:

  • 6 clones picked; 4 ml LB medium w/ 4 µl Cam each
  • Incubation at 37 °C over night

Picking of Ligations F188+F268 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES_eCFP_G_TEV-site_G_SYFP2), F230+F265 (PppActin_SERK-SigP_SpyCatcher_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_NanoLuc_SpyTag_t35S_npt-cassette), F230+F266 (PppActin_SERK-SigP_SpyTag_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_SpyCatcher_NanoLuc_t35S_npt-cassette) and F230+F267 (PppActin_SERK-SigP_Laccase_Strep-tag-II-TEV-site-Linker_SERK-TMD_8AALinker_GFPmut1_t35S_npt-cassette)

Investigator: Jeff

Aim of the experiment: Picking of Ligations F188+F268, F230+F265 (from 08/25 and from 08/26), F230+F266 (from 08/25 and from 08/26) and F230+F267 (from 08/25 and from 08/26)

Procedure:

  • 6 clones F188+F268
  • 3 clones each of F230+F265, F230+F266 and F230+F267 from 08/25
  • 3 clones each of F230+F265, F230+F266 and F230+F267 from 08/26
  • 4 ml LB medium w/ 4 µl Cam each
  • Incubation at 37 °C over night

Miniprep of P6 (pGAL in pTUM100)

Investigator: Jeff

Aim of the experiment: Preparation of P6 plasmid DNA

Procedure:

Preparation according to QIAGEN Miniprep Kit, elution in 50 µl EB buffer each

plasmid c (ng/µl)
P810 384.2
P811 134.1

Preparative digestion of P810 and P811 (pGAL in pTUM100) with with XbaI and PstI + Preparative Gelelectrophoresis + Gelextraction

Investigator: Jeff, Ingmar

Procedure:

Reaction batch for P810 and P811

volume reagent
25 µl Plasmid DNA
5 µl NEB 3.1 Buffer (10x)
1 µl XbaI (20 U/µl)
1 µl PstI (20 U/µl)
18 µl ddH2O
=50 µl TOTAL
  • Incubation at 37 °C for 2.5 h.
  • Preparative gelelectrophoresis at 90 V for 30 min on 1% agarose gel.

TUM13 20130827 prepVerd P810 P811 XbaI.PstI.png

  • Lowest band from digested P810 and P811 was cut out.
  • Gelextraction was performed with QIAquick Gel Extraction Kit, QIAGEN.

Ligation of F230+F273, F230+F274, F230+F275, F276+F277

Investigator: Jeff

Aim of the experiment: Ligation of F230+F273, F230+F274, F230+F275, F276+F277.

Procedure:

  • Ligation batch for F230+F273, vector:insert = 1:3
volume reagent
0.40 µl F230 (243.7 ng/µl; 5796 bp)
2.2 µl F273 (31.7 ng/µl; 1327 bp)
14.4 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F230+F274, vector:insert = 1:3
volume reagent
0.40 µl F230 (243.7 ng/µl; 5796 bp)
1.8 µl F274 (46.5 ng/µl; 1597 bp)
14.8 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F230+F275, vector:insert = 1:3
volume reagent
0.40 µl F230 (243.7 ng/µl; 5796 bp)
2.1 µl F275 (166.9 ng/µl; 6751 bp)
14.5 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F276+F277
volume reagent
4.3 µl F276 (243.7 ng/µl; 3978 bp)
12.7 µl F277 (166.9 ng/µl; 72 bp)
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • For neg. ctrl. water was used instead of insert.
  • Ligation was performed at room temperature for 1 h and then put on 16 °C for the next shift to transform the ligation products.

Wednesday, August 28th

Transformation of E. coli XL1-Blue with ligation products F230+F273 (Ig-Kappa-SigP_EreB + Plasmid with Mini-MCS), F230+F274 (IgKappa-SigP_Laccase + Plasmid with Mini-MCS), F230+F275 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES_eCFP_G_TEV-site_G_SYFP2 + Plasmid with Mini-MCS) and F276+F277 (pBad + digested pGAL)

Investigator: Louise

Aim of the experiment:Transformation of E. coli XL1-Blue with ligation products F230+F273 (Ig-Kappa-SigP_EreB + Plasmid with Mini-MCS), F230+F274 (IgKappa-SigP_Laccase + Plasmid with Mini-MCS), F230+F275 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES_eCFP_G_TEV-site_G_SYFP2 + Plasmid with Mini-MCS) and F276+F277 (pBad + digested pGAL).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 90 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates (having F230 as backbone) and kanamycin plates (having pBad as backbone).

Miniprep of ligation products F265+F230, F228+F230, F267+F230, F266+F230 and F268+F188

Investigator: Louise

Aim of the experiment:

Procedure:

  • Preparation according to QIAGEN Miniprep Kit, elution in 50 µl EB buffer each
  • The Plasmids were labelled as following:
ligation product label
F265+F230 clones 1a-3b P812-P817
F268+F188 clones 1-6 P818-P823
F228+F230 clones 1-5 P824-P828
F267+F230 clones 1a-3b P829-P834
F266+F230 clones 1a-3b P835-P840

Analytical digestion and gelelectrophoresis of P812-P840

Investigator: Louise

Aim of the experiment:Analytical digestion and gelelectrophoresis of P812-P840 .

Procedure:

  • Batch for digestion with XbaI and PstI
volume reagent
2.5 µl Plasmid DNA
2.0 µl NEBuffer 3.1
0.25 µl XbaI
0.25 µl PstI
15 µl ddH2O
=210 µl TOTAL


  • Analytical digestion was performed at 37 °C for 1 h.
  • After incubation 2  µl of DNA loading buffer (10x) were added to the reaction batches and 10 µl were loaded on a 1% agarose gel.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.


TUM13 20130828 analgel P812-P822 XbaI.PstI.png

Lane: 2 log DNA ladder P812 digested with XbaI & PstI P813 digested with XbaI & PstI P814 digested with XbaI & PstI P815 digested with XbaI & PstI P816 digested with XbaI & PstI P817 digested with XbaI & PstI P818 digested with XbaI & PstI P819 digested with XbaI & PstI P820 digested with XbaI & PstI P821 digested with XbaI & PstI P822 digested with XbaI & PstI
Result: TBD TBD TBD TBD TBD TBD TBD TBD TBD TBD TBD


TUM13 20130828 analgel P823-P833 XbaI.PstI 2.png

Lane: P823 digested with XbaI & PstI 2 log DNA ladder P824 digested with XbaI & PstI P825 digested with XbaI & PstI P826 digested with XbaI & PstI P827 digested with XbaI & PstI P828 digested with XbaI & PstI P829 digested with XbaI & PstI P830 digested with XbaI & PstI P831 digested with XbaI & PstI P832 digested with XbaI & PstI P833 digested with XbaI & PstI
Result: TBD TBD TBD TBD TBD TBD TBD TBD TBD TBD TBD


TUM13 20130828 analgel P834-P840 XbaI.PstI.png

Lane: P834 digested with XbaI & PstI 2 log DNA ladder P835 digested with XbaI & PstI P836 digested with XbaI & PstI P837 digested with XbaI & PstI P838 digested with XbaI & PstI P839 digested with XbaI & PstI P840 digested with XbaI & PstI
Result: TBD TBD TBD TBD TBD TBD TBD

Thursday, August 29th

Midiprep cultures from Miniprep cultures P815 and P835 as well as P823-P828

Investigator: Jeff, Ingmar

Aim of the experiment: Midiprep culture from Miniprep cultures P815 (SERK-SigP_SpyCatcher_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_NanoLuc_SpyTag + Plasmid mit Mini MCS), P835 (SERK-SigP_SpyTag_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_SpyCatcher_NanoLuc + Plasmid mit Mini MCS) and P823-P828 (Banana odor generator in Mini MCS)

Procedure:

  • 50 ml LB medium w/ 50 µl chloramphenicol stock (1:1000)
  • 1 ml preculture each

Picking of Ligation F276+F277 (pBad + MCS w/ StrepTag)

Investigator: Jeff

Procedure:

  • 2 clones picked; 4 ml LB medium w/ 8 µl Kanamycin
  • Incubation at 37 °C

Preparative digestion of P823 with EcoRI & PstI and gelextraction

Investigator: Jeff

Aim of the experiment: Preparative digestion of P823 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + eCFP_G_TEV-site_G_SYFP2)

Procedure:

Reaction batch for P823

volume reagent
20 µl Plasmid DNA
4 µl NEB 2.1 Buffer (10x)
2 µl EcoRI-HF(20 U/µl)
2 µl PstI (20 U/µl)
12 µl ddH2O
=40 µl TOTAL
  • Incubation at 37 °C for 1.5 h.
  • Preparative gelelectrophoresis at 90 V for 30 min on 1% agarose gel.

TUM13 20130829 prepVerd P823 EcoRI.PstI.png

  • Result as expected; the upper band was cut out, processed with QIAGEN gel extraction kit and labeled F279

Picking of Retrafos of P290, P387, P787, P794, P799

Investigator: Rosario

Aim of the experiment:

Picking of Retrafos of P290, P387, P787, P794, P799

Procedure:

  • 2 clones picked; 4 ml LB medium w/ 4 µl Cam each (Kan for P799)
  • Incubation at 37 °C over day

Picking of Ligation products F230+F273, F230+F274, F230+F275 and F276+F277

Investigator: Louise

Aim of the experiment: Picking of Ligation products F230+F273, F230+F274, F230+F275 and F276+F277


Procedure:

  • The picked clones were each transferred into 4 ml LB medium with 4 µl Chloramphenicol (Kan for F276+F277)
  • Incubation at 37 
Ligation amount of clones picked
F230+F273 4
F230+F274 4
F230+F275 2
F276+F277 2

Transformation of E. coli XL1-Blue with ligation product F230+F279

Investigator: Louise

Aim of the experiment:Transformation of E. coli XL1-Blue with ligation product F230+F279.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 90 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol (for P713 kanamycin) plates.


Miniprep of ligation products F268+F187 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + eCFP_G_TEV-site_G_SYFP2) and F268+F188 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + eCFP_G_TEV-site_G_SYFP2)

Investigator: Ingmar

Aim of the experiment:

Procedure:

  • Preparation according to QIAGEN Miniprep Kit, elution in 50 µl EB buffer each
  • The Plasmids were labelled as following:
ligation product label
F268+F187 clones 1-3 P841-P843
F268+F188 clones 1-3 P844-P846

Analytical digestion and gelelectrophoresis of P841-P846

Investigator: Ingmar

Aim of the experiment:Analytical digestion and gelelectrophoresis of P841-846 .

Procedure:

  • Batch for digestion with XbaI and PstI
volume reagent
2.5 µl Plasmid DNA
2.0 µl NEBuffer 3.1
0.25 µl XbaI
0.25 µl PstI
15 µl ddH2O
=20 µl TOTAL


  • Analytical digestion was performed at 37 °C for 1 h.
  • After incubation 2  µl of DNA loading buffer (10x) were added to the reaction batches and 10 µl were loaded on a 1% agarose gel.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.

TUM13 20130829 analgel P841-P8465 XbaI.PstI.png

Lane: 2 log DNA ladder P841 digested with XbaI & PstI P842 digested with XbaI & PstI P843 digested with XbaI & PstI P844 digested with XbaI & PstI P845 digested with XbaI & PstI P846 digested with XbaI & PstI
Result: correct correct correct false false false

Picking of E. coli XL1-Blue transformed F230+F279 (PppActin_PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES_eCFP_G_TEV-site_G_SYFP2_t35S_npt-cassette)

Investigator: Jeff

Aim of the experiment: Picking of E. coli XL1-Blue transformed F230+F279 (PppActin_PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES_eCFP_G_TEV-site_G_SYFP2_t35S_npt-cassette).

Procedure:

  • 6 colonies were picked from the plates with E. colis transformed with the ligation product F230+F279.
  • Picked colonies were transferred in sterile air-permeable cell culture tubes with 4 µl LB medium with chloramhpenicol.
  • Incubation at 37 °C overnight.

Miniprep of ligation products F230+F273, F230+F274, F230+F275, F230+F277 and of P290, P387, P787, P794, P799

Investigator: Rosario

Aim of the experiment: Miniprep of ligation products F230+F273, F230+F274, F230+F275, F230+F277 and of P290, P387, P787, P794, P799.

Procedure:

Miniprep according to QIAGEN QIAprep Kit Tubes were labelled P847 - P858.

Friday, August 30th

Miniprep of F276 + F277 (pBAD + MCS) and F230 + F279 (FRET in pActin_npt-cassette)

Investigator: Florian

Aim of the experiment: Miniprep of F276 + F277 (pBAD + MCS) and F230 + F279 (FRET in pActin_npt-cassette).

Procedure:

Miniprep according to QIAGEN QIAprep Kit

Analytical digestion and gelelectrophoresis of P857 and P858

Investigator: Florian

Aim of the experiment: Analytical digestion and gelelectrophoresis of P857 and P858.

Procedure:

  • Batch for digestion with XbaI.
volume reagent
2.5 µl Plasmid DNA
2.0 µl NEBuffer CutSmart
0.25 µl XbaI
15.25 µl ddH2O
=20 µl TOTAL


  • Analytical digestion was performed at 37 °C for 1 h.
  • After incubation 2  µl of DNA loading buffer (10x) were added to the reaction batches and 20 µl were loaded on a 1% agarose gel.
  • Analytical gelelectrophoresis was performed at 90 V for ?? min.

TUM13 20130830 anal Verd F276 P857-P858 XbaI.png

Lane: 2 log DNA ladder F276 P857 digested with XbaI P858 digested with XbaI
Result: inconclusive inconclusive

Analytical digestion and gelelectrophoresis of P868 and P869

Investigator: Florian

Aim of the experiment: Analytical digestion and gelelectrophoresis of P868 and P869.

Procedure:

  • Batch for digestion with XbaI.
volume reagent
2.5 µl Plasmid DNA
2.0 µl NEBuffer CutSmart
0.25 µl XbaI
15.25 µl ddH2O
=20 µl TOTAL


  • Analytical digestion was performed at 37 °C for 1 h.
  • After incubation 2  µl of DNA loading buffer (10x) were added to the reaction batches and 20 µl were loaded on a 1% agarose gel.
  • Analytical gelelectrophoresis was performed at 90 V for ?? min.

TUM13 20130830 anal Verd F276 P868-P869 XbaI.png

Lane: 2 log DNA ladder F276 P868 digested with XbaI P869 digested with XbaI
Result: inconclusive as expected

Analytical digestion and gelelectrophoresis of P870 - P875

Investigator: Florian

Aim of the experiment: Analytical digestion and gelelectrophoresis of P870 - P875.

Procedure:

  • Batch for digestion with XbaI and PstI.
volume reagent
2.5 µl Plasmid DNA
2.0 µl NEBuffer 3.1
0.25 µl XbaI
0.25 µl PstI
15 µl ddH2O
=20 µl TOTAL


  • Analytical digestion was performed at 37 °C for 1 h.
  • After incubation 2  µl of DNA loading buffer (10x) were added to the reaction batches and 20 µl were loaded on a 0.5% agarose gel.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.

TUM13 20130830 anal Verd P870-P875 XbaI.PstI.png

Lane: 2 log DNA ladder P870 digested with XbaI & PstI P871 digested with XbaI & PstI P872 digested with XbaI & PstI P873 digested with XbaI & PstI P874 digested with XbaI & PstI P875 digested with XbaI & PstI
Result: as expected bad bad as expected bad bad

Preparative digestion of P717 (Laccase-rec.) and P860 (EreB-sec.) + Preparative Gelelectrophoresis + Gelextraction

Investigator: Florian

Aim of the experiment: Preparative digestion of P717 (Laccase-rec.) and P860 (EreB-sec.) + Preparative Gelelectrophoresis + Gelextraction.


Procedure:

Reaction batch for P717:

volume reagent
20 µl P717
4 µl 2.1 Buffer (10x)
1.5 µl EcoRI (20 U/µl)
2 µl PstI (20 U/µl)
3 µl MlyI (10 U/µl)
9.5 µl ddH2O
=40 µl TOTAL

Reaction batch for P860:

volume reagent
20 µl P860
4 µl 2.1 Buffer (10x)
1.5 µl EcoRI (20 U/µl)
2 µl PstI (20 U/µl)
12.5 µl ddH2O
=40 µl TOTAL
  • Reaction batches were incubated at 37 °C for 3 h.
  • Preparative gelelectrophoresis was performed at 90 V for 1 h in an 0.5% agarose gel.

TUM13 20130830 prep Verd P717 EcoRI.PstI.MlyI P860 EcoRI.PstI.png

Lane 2log DNA ladder Digestion of P717 with EcoRI & PstI & MlyI Digestion of P860 with EcoRI & PstI
Results as expected; upper band was cut out as expected; lower band was cut out
  • Gelextraction was performed after preparative Gelelectrophoresis
  • The resulting fragments were named F280 (Laccase-rec.) + F281 (EreB-sec.)

Ligation of F230+F280 (Laccase-rec.) and F230+F281 (EreB-sec.)

Investigator: Jeff, Florian

Aim of the experiment: Ligation of F230+F280 (Laccase-rec.) and F230+F281 (EreB-sec.).

Procedure:

  • Ligation batch for F230+F280, vector:insert = 1:3
volume reagent
0.416 µl F230 (243.7 ng/µl; 5796 bp)
3.314 µl F280 (41.1 ng/µl; 2632 bp)
13.3 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F230+F281, vector:insert = 1:3
volume reagent
0.416 µl F230 (243.7 ng/µl; 5796 bp)
1.659 µl F281 (41.4 ng/µl; 1327 bp)
14.93 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • For neg. ctrl. water was used instead of insert.
  • Ligation was performed at room temperature for 1 h and then transformed.

Transformation of E. coli XL1-Blue with ligation product F230+F280 (Laccase-rec.) and F230+F281 (EreB-sec.)

Investigator: Flo

Aim of the experiment:Transformation of E. coli XL1-Blue with ligation product F230+F280 (Laccase-rec.) and F230+F281 (EreB-sec.).

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 90 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Sunday, September 01th

Preparative digestion of P624 (GST) and P645 (Banana) + Preparative Gelelectrophoresis + Gelextraction

Investigator: Andi

Aim of the experiment: Preparative digestion of P624 (GST) and P645 (Banana) + Preparative Gelelectrophoresis + Gelextraction.


Procedure:

Reaction batch for P624:

volume reagent
20 µl P624
4 µl Cut Smart Buffer
1 µl XbaI (20 U/µl)
1 µl PstI (20 U/µl)
14 µl ddH2O
=40 µl TOTAL

Reaction batch for P645:

volume reagent
20 µl P645
4 µl Cut Smart Buffer
1 µl XbaI (20 U/µl)
1 µl PstI (20 U/µl)
14 µl ddH2O
=40 µl TOTAL
  • Reaction batches were incubated at 37 °C for 2.5 h.
  • Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.

500px

Lane 1kB DNA ladder Digestion of P624 with XbaI and PstI Digestion of P645 with XbaI and PstI
Results as expected; upper band was cut out as expected; lower band was cut out
  • Gelextraction was performed after preparative Gelelectrophoresis
  • The resulting fragments were named FBLUB (GST) and FBLUB (BANANE)

PCR, purification and analytical geleletrophoresis of P634 (pActin) and P800 (pAct_MCS)

Investigator: Rosario

Aim of the experiment: PCR, purification and analytical geleletrophoresis of P554 (pActin) and P800 (pAct_MCS).

Procedure:

Operational sequence:

  • PCR reaction mixture
volume reagent
25 µl 2x Q5 Master Mix
2.5 µl 10 µM Forward Primer O126 (pAct5_fw_cor)
2.5 µl 10 µM Reverse Primer O117 (pAct5_rv)
1 µl Plasmid DNA (P634) - (desired c between 1 ng and 5 ng)
19 µL ddH2O Water
=50 µL TOTAL
volume reagent
25 µl 2x Q5 Master Mix
2.5 µl 10 µM Forward Primer O126 (pAct5_fw_cor)
2.5 µl 10 µM Reverse Primer O117 (pAct5_rv)
1 µl Plasmid DNA (P800) - (desired c between 1 ng and 5 ng)
19 µL ddH2O Water
=50 µL TOTAL
  • Mix with pipette
Initial denaturation 98 °C 30 s
30 cycles 98 °C 30 s
54 °C; 60 s
72 °C 2 min
Final extension 72 °C 2 min
Hold 4 °C infinite
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
  • 9 µl of the purified PCR products were mixed with 1 µl DNA loading buffer (10x) for analytical gelelectrophoresis
  • Analytical gelelectrophoresis was performed at 90 V for 60 min in 1% agarose gel.
1 kbp ladder Fragment of digested P634 Fragment of digested P800
Fragment-size like expected also pppActin5 without MCS

TUM13 20130901 F282 P283 pcr.png

Week 19

Tuesday, September 03th

Ligation of F276+F289 and F276+F290

Investigator: Andi

Aim of the experiment: Ligation of F276+F289 and F276+F290.

Procedure:

  • Ligation batch for F276+F289, vector:insert = 1:3
volume reagent
4.3 µl F276
6.5 µl F280
6.2 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation batch for F276+F290, vector:insert = 1:3
volume reagent
4.3 µl F276
12.7 µl F290
0 µl ddH2O
2 µl T4 ligase buffer (10x)
1 µl T4 ligase (1 U/µl)
=20 µl TOTAL
  • Ligation was performed at room temperature for 1 h and then transformed.

Transformation of E. coli XL1-Blue with ligation product F289+F276 and F290+F276

Investigator: Andi

Aim of the experiment:Transformation of E. coli XL1-Blue with ligation product F289+F276 and F290+F276

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 90 min at 37 °C in the 180 rpm cell-culture shaker.
  • The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new Kanamycin plates.

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