Team:TU-Munich/Notebook/Labjournal

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Revision as of 04:44, 29 August 2013 by Leonie (Talk | contribs)

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Labjournal

Display:
General
Vector Design
Effectors
Vitality Sensor
Safety
Expand All ...
Collapse All ...
Jump to:
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

[Expand] 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.

[Expand] 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)

[Expand] 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

[Expand] 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

[Expand] 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
  • Batch for analytical digestion for P5 with NgoMIV+AgeI-HF
  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.

Results:

  • Parts are compliant and do not contain RFC25 forbidden restriction sites.


TUM13 20130423 RFP Generator RFC25 AgeI NgoMIV.png

[Expand] 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

[Expand] 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)

[Expand] 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
  • Batch for analytical digestion for P8 with NgoMIV+AgeI-HF
  • Batch for analytical digestion for P9 with NgoMIV+AgeI-HF
  • Batch for analytical digestion for P10 with NgoMIV+AgeI-HF
  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.

Results:


TUM13 20130424 PhytochromeB RFC25 AgeI NgoMIV.png

[Expand] 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

[Expand] 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:
  • The procedure will have to be repeated for pRIL

[Expand] 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

[Expand] 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
  • Batch for preparative digestion for Leptin with HindIII and EheI
  • Incubation for 3 h at 37 °C.

[Expand] 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

[Expand] 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

[Expand] 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.

[Expand] 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:

Sunday, May 5th

[Expand] 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:
  • 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

[Expand] 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:


[Expand] 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:


[Expand] 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:

Tuesday, May 7th

[Expand] 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
  • 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)):
  • 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.

TUM13 20130507 P15 P16 PCR.png

[Expand] 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
  • Batch for preparative digestion of 20aalinker-PIF3 (P51) with EcoRI and NgoMIV
  • 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.

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

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

Wednesday, May 8th

[Expand] 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.
  • Batch for preparative digestion of P52 for preperation of pSB1C3 vector:
  • 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.

TUM13 20130508 F1 F2 P52 Xb.png

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

[Expand] 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
  • Ligation batch for F8+F7
  • 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:


Lid temperature = 37 °C

[Expand] 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

[Expand] 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

[Expand] 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

Reaction batch 2


PCR cycling parameters

  • 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

[Expand] 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)

[Expand] 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
  • Mix with pipette
  • The PCR program was performed after following scheme with following conditions:
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
  • Resulting product was labeled as F12.

[Expand] Digestion of P61 with S1 Nuclease

Investigator: Katrin

[Expand] 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

[Expand] 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:

Saturday, May 11th

[Expand] 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.
  • 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

[Expand] 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

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


TUM13 20130511 P63 P64 XbaI.png

[Expand] 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.
  • Incubation for 3 h at 37 °C
  • The digested fragment was labelled F14

[Expand] 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)
  • 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:


Lid temperature = 37 °C

[Expand] 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

[Expand] 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

[Expand] 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

Reaction batch 2

PCR cycling parameters

  • 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

[Expand] 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

[Expand] 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.

[Expand] 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.

[Expand] 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.

[Expand] 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

[Expand] 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)

[Expand] 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

[Expand] 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

Reaction batch 2

PCR cycling parameters

  • 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.

[Expand] 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

Reaction batch 2

PCR cycling parameters

  • 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.

[Expand] 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

Reaction batch 2

PCR cycling parameters

  • 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.

[Expand] 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.

[Expand] 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.

[Expand] 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

[Expand] 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

Batch of Mastermix

  • 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:
  • Gel 1, Row 2:
  • Gel 2:

[Expand] 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.

[Expand] 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:


Wednesday, May 15th

[Expand] 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.
  • Incubation for 2.5 h at 37 °C
  • The digested fragments were labelled F17(P8), F18(P60), F19(P64)

[Expand] 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
  • Mix with pipette
  • The PCR program TMKS was performed after following scheme:
  • 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.

TUM13 20130515 PCR P16.png

[Expand] 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)
  • 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:

[Expand] 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:
  • Batch for digestion of P90-P96 with XbaI+AgeI-HF:
  • Batch for digestion of P90-P96 with XbaI+AgeI-HF:
  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.

Results:

  • 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

[Expand] 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
  • Batch for preparative digestion of psB1A2 with EcoRI
  • Batch for preparative digestion of P96 (TEV S219V) with XbaI & AgeI.
  • 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.

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

[Expand] 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.
  • Incubation for 2.5 h at 37 °C
  • The digested fragments were labelled

[Expand] 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
  • 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

[Expand] 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)


  • Ligation batch for TEV (F20) with pSB1C3 (F17)
  • Ligation batch for pSH21 (F22) with pSB1A2(F23)
  • Ligation batch for EreA (F19) with pSB1C3 (F17)


  • 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

[Expand] 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)

[Expand] 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



PCR cycling parameters

[Expand] 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

[Expand] 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

[Expand] 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)

[Expand] 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:
  • Batch for digestion of F22+F23 with EcoRI-HF:
  • 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 20130519 P98-P103 XbaI.PstI P104-P105 EcoRI(1).png

[Expand] 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

Reaction batch 2

PCR cycling parameters

  • 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.

[Expand] 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

[Expand] 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

[Expand] 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

Reaction batch 2

PCR cycling parameters

  • 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.

[Expand] 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

[Expand] 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.

[Expand] 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:

[Expand] 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

[Expand] 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

[Expand] 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)

[Expand] 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.

[Expand] 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:

  • 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:

  • 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:

  • 17,5 µl Mastermix with 2,5 µl Plasmid


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

  • 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


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


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

Thursday, May 23rd

[Expand] 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:

[Expand] 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
  • Mix with pipette
  • The PCR program was performed after following scheme with following conditions:
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
  • Resulting product was labeled as F24 (N-TEV) and F25 (C-TEV).

[Expand] 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.

[Expand] 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.
  • 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)

[Expand] 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:
  • Ligation batch for F17+F27 (positive control)
  • 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:
  • Lid temperature = 37 °C

[Expand] 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)

[Expand] 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:
  • 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

[Expand] 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


PCR cycling parameters - P106

  • 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)


PCR cycling parameters - P116

  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

[Expand] 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

[Expand] 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.

[Expand] 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


PCR cycling parameters - P109

  • 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)


PCR cycling parameters - P116

  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

[Expand] 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

[Expand] 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.


[Expand] 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.

[Expand] 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

[Expand] 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)

[Expand] 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)
  • 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)
  • 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

Laccase P124 was chosen for sequencing


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

EreB P125 was chosen for sequencing

[Expand] 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)

[Expand] 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:


  • 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

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

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

[Expand] 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:

Week 6

Monday, May 27th

[Expand] 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

[Expand] 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
  • Mix with pipette
  • The PCR program was performed after following scheme:
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

Results:

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

[Expand] 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.

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

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

  • 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

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

[Expand] 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

Reaction batch - P125

PCR cycling parameters for both batches

  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

[Expand] 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:

[Expand] 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.

[Expand] 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.

[Expand] 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:
  • Ligation batch for F17+F28:
  • 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:
  • Lid temperature = 37 °C

Tuesday, May 28th

[Expand] 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

  • Wrong buffer (GC buffer, instead of standard reaction buffer, was used and 10x too much primers were also used)

[Expand] 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.

[Expand] 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:

[Expand] 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)

[Expand] 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.

[Expand] 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
  • Mix with pipette
  • The PCR program was performed after following scheme:
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

[Expand] 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

Wednesday, May 29th

[Expand] 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.

[Expand] 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)

[Expand] 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.

[Expand] 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


PCR cycling parameters - P133

  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

[Expand] 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.

[Expand] 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
  • Batch for preparative digestion of npt(F35) with EcoRI and SpeI
  • Batch for preparative digestion of PIF3(F36) with XbaI and AgeI-HF
  • Batch for preparative digestion of P10 with XbaI & PstI for backbone isolation.
  • Batch for preparative digestion of P10 with EcoRI and SpeI for backbone isolation.
  • 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


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

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

[Expand] 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.

[Expand] PCR of pActin (P143)

Investigator: Florian, Jeff

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

Procedure:

Operational sequence:

  • PCR reaction mixture
  • Mix with pipette
  • The PCR program was performed after following scheme:
  • 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!

[Expand] 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:
  • Ligation batch for F42+F38:
  • Ligation batch for F41+F39:
  • 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:
  • Lid temperature = 37 °C

Thursday, May 30th

[Expand] Gradient PCR of pActin (P143)

Investigator: Ingmar

Aim of the experiment: PCR of pActin

Procedure:

Operational sequence:

  • PCR reaction mixture
  • PCR reaction mixture
  • Mix with pipette
  • The PCR program was performed after following scheme:

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

[Expand] 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)

[Expand] 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.
  • 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

[Expand] 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

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

[Expand] 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.

[Expand] 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:
  • Ligation batch for F17+F32:
  • 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:
  • Lid temperature&

[Expand] 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

[Expand] 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)

[Expand] 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
  • 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

[Expand] 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:

[Expand] Ligation of F29+F30

Investigator: Katrin

Aim of the experiment: Ligation of F29+F30

Procedure:

  • Ligation batch


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

[Expand] 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


PCR cycling parameters - P124

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

[Expand] 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
  • 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

[Expand] Ligation of F42+F50 (pActin in pSB1C3)

Investigator: Ingmar

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

Procedure:

  • Ligation batch
  • 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:
  • Lid temperature = 37 °C

[Expand] 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.

[Expand] 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

[Expand] 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.

[Expand] 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.

[Expand] 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)

[Expand] 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
  • 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
  • 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

Sunday, June 2nd

[Expand] 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

[Expand] 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.
  • 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):
  • 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


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

Tuesday, June 4th

[Expand] 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:

[Expand] 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
  • Batch for preparative digestion of P20(GFP-Psb1C3) with NgoMIV and PstI
  • Batch for preparative digestion of F45(pActin) with XbaI and PstI
  • 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

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

[Expand] 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


PCR cycling parameters - P179

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

[Expand] 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
  • Batch for preparative digestion of P183 with XbaI and AgeI- HF
  • 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

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

[Expand] 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
  • 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.

[Expand] 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.

[Expand] 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.

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

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.

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

  • Incubation for 150 min at 37 °C.
  • Preparative gelelectrophoresis was performed at 90 V for 1 h.

File:FOTOFOTO.png

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

[Expand] 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 :
  • Ligation batch for F55+F59:
  • Ligation batch for F56+F59:
  • Ligation batch for F57+F59:
  • Ligation batch for F58+F59:
  • Negative Control Ligation batch for F59:
  • Ligation batch for F52+F53:
  • Ligation batch for F42+F51:
  • 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:
  • Lid temperature = 37 °C

[Expand] 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:

Wednesday, June 5th

[Expand] 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.

[Expand] 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

[Expand] 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:

[Expand] 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:

Mastermix (12x)

  • 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

[Expand] 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.

[Expand] 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
  • Mix with pipette
  • The gradient PCR program was performed after following scheme with following conditions:
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
  • PCR product of P192 = F61; PCR product of 194 = F62

[Expand] 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


PCR cycling parameters - P179

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

[Expand] 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

[Expand] Ligation of F42+F51

Investigator: Ingmar

Aim of the experiment: Ligation of F42+F51.

Procedure:

  • Ligation batch for F42+F51:
  • 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

[Expand] 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).

[Expand] 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):



The different genes received the following barcodes:

[Expand] 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:
  • Batch for preparative digestion of F62 (PIF6) with XbaI & AgeI:
  • Batch for preparative digestion of P165 (t35S) with XbaI:
  • Batch for preparative digestion of F31 (miniMCSII):
  • 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


TUM13 20130606 prep Verd P165 XbaI.png


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

[Expand] 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
  • 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

[Expand] 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):
  • Ligation batch for F59+F66:
  • Ligation batch for F63+F64:
  • 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

[Expand] 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.

[Expand] 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

[Expand] 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:


[Expand] 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).

[Expand] 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)

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:

[Expand] 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
  • 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



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

Saturday, June 8th

[Expand] 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:

[Expand] 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.


[Expand] 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
  • Batch for preparative digestion of P202 (GFP+linker, fragment named F69) with NgoMIV/SpeI
  • Batch for preparative digestion of P210 (SERK-TMD, fragment named F67) with AgeI/SpeI


  • 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


TUM13 20130608 prep Verd P202 NgoMIV.SpeI.png



TUM13 20130608 prep Verd P210 AgeI.SpeI.png



  • Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
  • Concentration determined with Nanodrop:

[Expand] 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
  • Batch for preparative digestion of P8 (PhyB in pSC1C3, fragment named F76) with NgoMIV/SpeI
  • 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


  • Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
  • Concentration determined with Nanodrop:



[Expand] 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
  • 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

[Expand] 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
  • Batch for preparative digestion of P198 (gene synthesis 2, fragments named F72-F74) with XbaI/AgeII
  • 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


  • Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
  • Concentrations measured with Nano-Drop


[Expand] 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

[Expand] 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:


  • Incubation: 1 h 22°C


[Expand] 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

[Expand] 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:

[Expand] 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
  • 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


  • 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


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

[Expand] 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)

[Expand] 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
  • Batch for preparative digestion of P160 (NLS, fragment named F77) with AgeI/SpeI
  • Batch for preparative digestion of P40 (GSAT linker, fragment named F79) with NgoMIV/SpeI
  • Batch for preparative digestion of P7 (PhyB, fragment named F78) with AgeI/SpeI
  • 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

  • the band was extracted with QIAquick Gel Extraction Kit, QIAGEN.

[Expand] 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

[Expand] 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)
  • Ligation batch for F59+F71 (pSB1C3+Ig-K)
  • 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.

[Expand] 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).

[Expand] PCR of P192 (Thermonuclease, O64 & O65)

Investigator: Katrin

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

Procedure:

Operational sequence:

  • PCR reaction mixture
  • Mix with pipette
  • The gradient PCR program was performed after following scheme with following conditions:

Week 8

Monday, June 10rd

[Expand] 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

[Expand] 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

[Expand] 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

[Expand] 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.

[Expand] 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).

[Expand] 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
  • Mix with pipette
  • The PCR program was performed after following scheme:
  • 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

  • As we can see, the fragment has the expected length of about 1600 BP.

[Expand] 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.
  • 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

[Expand] 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)
  • 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.

[Expand] 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).

[Expand] 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
  • 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)

[Expand] 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)
  • 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

[Expand] 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.


[Expand] 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:

[Expand] 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
  • 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


TUM13 20130611 anal gel P239-P241 XbaI AgeI.png


[Expand] 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
  • Batch for preparative digestion of P157 (EreB, fragment named F86) with NgoMIV/SpeI
  • Batch for preparative digestion of P208 (NanoLuc, fragment named F87) with NgoMIV/SpeI
  • Batch for preparative digestion of P213 (Spycatcher, fragment named F88) with NgoMIV/SpeI
  • Batch for preparative digestion of P170 (Spytag, frament named F89) with NgoMIV/SpeI
  • 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

20130611 prep Verd P208, P1213 NgoMIV.SpeI.png

20130611 prep Verd P170 AgeI.SpeI.png

  • the band was extracted with QIAquick Gel Extraction Kit, QIAGEN.

[Expand] 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)
  • Ligation batch for F90+F87 (IgKappa-SigP + Nanoluciferase)
  • Ligation batch for F90+F88 (IgKappa-SigP + SpyCatcher)
  • Ligation batch for F90+F89 (IgKappa-SigP + SpyTag)
  • 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.

[Expand] 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
  • Second batch - PCR reaction mixture
  • Third batch - PCR reaction mixture
  • The content of all batches has been mixed with a pipette
  • The PCR program was performed after following scheme:


  • The PCR for P192(Thermonuclease) was performed three times under different conditions
  • First batch - PCR reaction mixture
  • Second batch - PCR reaction mixture
  • Third batch - PCR reaction mixture
  • The PCR program of the Thermonuclease (P192) was performed after following scheme:
  • 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
  • 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

  • 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.


[Expand] 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.
  • 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.
  • 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

[Expand] 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)
  • Ligation batch for F85+F59 (psB1C3+Thermonuclease)
  • 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.

[Expand] 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

[Expand] 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.

[Expand] 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
  • Batch for preparative digestion of P244 (IgKappa) with AgeI/SpeI
  • Batch for preparative digestion of P197 (XylE) with NgoMIV/SpeI
  • Batch for preparative digestion of P150 (FluA) with NgoMIV/SpeI
  • Batch for preparative digestion of P126 (N-TEV) with AgeI/SpeI
  • Batch for preparative digestion of P127 (C-TEV) with NgoMIV/SpeI
  • Batch for preparative digestion of P168 (PIF3) with NgoMIV/SpeI
  • Batch for preparative digestion of P222 (PIF6) with NgoMIV/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 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

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

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

  • the DNA was extracted from the gel with QIAquick Gel Extraction Kit, QIAGEN.
  • the fragments received the following names:

[Expand] 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.

[Expand] 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.


[Expand] 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 :
  • Ligation batch for F92+F87 :
  • Ligation batch for F92+F86:
  • Ligation batch for F92+F88 :
  • Ligation batch for F92+F97:
  • Ligation batch for F90+F97:
  • Ligation batch for F91+F94:
  • Ligation batch for F93+F79:
  • 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

[Expand] 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

[Expand] 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)

Reaction batch for P163 (NPT-Cassette)


Reaction batch for P250 (Laccase)


Reaction batch for P217 (Laccase)

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

  • After the PCR, the Quickchange product was stored in the fridge.

[Expand] 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
  • PCR reaction mixture for IRES
  • 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)):
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
  • Resulting products was labeled as F99 (AlcR) and F100 (IRES).

[Expand] 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

[Expand] 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:

[Expand] 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
  • 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


[Expand] 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.

[Expand] 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).


[Expand] 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

[Expand] 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.

[Expand] 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:

[Expand] 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.

[Expand] 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:

[Expand] 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
  • 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

TUM13 20130614 anal Verdau P259-P265 EcoRI.SpeI.png

TUM13 20130614 anal Verdau P266-P272 EcoRI.SpeI.png

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


[Expand] Preparative Digestion of IRES (F100) and AlcR (F99) with EcoRI and SpeI

  • Incubation for 150 min at 37 °C
  • Purification by QIAGEN PCR Purification Kit
  • new Names: F99 -> F101 and F100 -> F102

[Expand] 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

[Expand] 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:

[Expand] 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:
  • Ligation batch for F41+F102 (IRES of poliovirus), 100 ng vector used, 3:1:
  • 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.

[Expand] 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
  • 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

[Expand] 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

[Expand] 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)

Reaction batch for P163 (NPT-Cassette)

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

  • After the PCR, the Quickchange product was stored in the fridge.

[Expand] 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.

[Expand] 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
  • Ligation batch for F59+F103 (Strep-TEV), 100 ng vector used, insert:vector = 3:1
  • Negative control was prepared by replacing the volume of the insert by ddH2O.
  • The ligation was performed for 1 hour at room temperature.


[Expand] 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

[Expand] 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:

[Expand] 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.

[Expand] 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
  • Batch for preparative digestion of P299 (IRES) with NgoMIV/SpeI
  • Batch for preparative digestion of P274 (nTevlinker)with AgeI/SpeI
  • Batch for preparative digestion of P111 (TEV) with AgeI/SpeI
  • Batch for preparative digestion of P248 (Nuclease) with NgoMIV/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 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

TUM13 20130617 prep Verd P274 AgeI.SpeI P299 NgoMIV.SpeI.png

  • The DNA was extracted from the gel with QIAquick Gel Extraction Kit, QIAGEN

[Expand] 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
  • 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

[Expand] 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.

[Expand] 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


[Expand] 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
  • Negative control was prepared by replacing the volume of the insert by ddH2O.
  • The ligation was performed overnight at 16 °C.

[Expand] 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.


[Expand] 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:

Tuesday, June 18th

[Expand] 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.

[Expand] 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.

[Expand] 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:

[Expand] 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
  • 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

[Expand] 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
  • Batch for preparative digestion of P238 (SERK-TMD_Linker_GFP) with NgoMIV/SpeI
  • Batch for preparative digestion of P303 (SERK-SigP) with AgeI/SpeI
  • Batch for preparative digestion of P304 (StrepTEV-Linker) with AgeI/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 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

TUM13 20130618 prep Verd P212 NgoMIV.SpeI.png

  • The DNA was extracted from the gel with QIAquick Gel Extraction Kit, QIAGEN

[Expand] 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
  • Ligation batch for F109+F87 (SERK-SigP + NanoLuc), 100 ng vector used, insert:vector = 3:1
  • Ligation batch for F109+F88 (SERK-SigP + SpyCatcher), 100 ng vector used, insert:vector = 3:1
  • Ligation batch for F109+F89 (SERK-SigP + SpyTag), 100 ng vector used, insert:vector = 3:1
  • Ligation batch for F109+F97 (SERK-SigP + XylE), 100 ng vector used, insert:vector = 3:1
  • Ligation batch for F109+F107 (SERK-SigP + SERK-TMD), 100 ng vector used, insert:vector = 3:1
  • Ligation batch for F110+F108 (Strep-tag-TEV-site-linker + SERK-TMD_8AAlinker_GFPmut1), 100 ng vector used, insert:vector = 3:1
  • Negative control was prepared by replacing the volume of the insert by ddH2O.
  • The ligation was performed for 1 hour at room temperature.

[Expand] 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)

Reaction batch for P163 (NPT-Cassette)

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

  • After the PCR, the Quickchange products were mixed with 1 µl of DpnI (10 U/µl) and incubated at 37 °C for 1 h.

[Expand] 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

[Expand] 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.

[Expand] 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:

Wednesday, June 19th

[Expand] 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)

PCR cycling parameters - P294 (AlcR)

  • After the PCR, the Quickchange products were mixed with 1 µl of DpnI (10 U/µl) and incubated at 37 °C for 1 h.

[Expand] 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

[Expand] 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.

[Expand] 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:

[Expand] 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:

[Expand] 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

[Expand] 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

[Expand] 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:

[Expand] 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).

[Expand] 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:

[Expand] 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
  • Batch for 500ml of a 50 mM CaCl2-solution
  • Batch for 50ml of a 50 mM CaCl2-solution, 15% v/v Glycerin
  • Autoclave each bottle of solution and leave each in a 4°C room.

[Expand] 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

PCR cycling parameters - P114 (pActin)

Reaction batch 2 for P114 (pActin) with Q5 Polymerase Mastermix

PCR cycling parameters - P114 (pActin) with Q5 Polymerase

Reaction batch for P294 (AlcR) with Pfu Ultra II

PCR cycling parameters - P294 (AlcR)

  • 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


[Expand] 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.


[Expand] 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
  • Reaction batch with Q5 Mastermix
  • The content has been mixed with a pipette
  • The PCR program was performed after following scheme:
  • After the PCR has finished, the product was hold at 4 °C in the Cycler

[Expand] 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

[Expand] 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
  • 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

TUM13 20130620 anal Verd P334 P335 P170 P336 P337 P197 P338 P339 P212 P320 PstI.EcoRI.png


TUM13 20130620 anal Verd P320 326 327 PstI.EcoRI.png

TUM13 20130620 anal Verd P328 P329 P157 P330 P331 P208 P332 P333 P213 P326 P327 PstI.EcoRI.png

Friday, June 21st

[Expand] 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

[Expand] 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

[Expand] 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)

  • 20 µl Mastermix + 20 µl Plasmid each

Batches for preparative digestions:

  • 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


TUM13 20130621 prep Verd. P326 NgoMIV .PstI P328 AgeI.Pst P330 AgeI.PstI.png

TUM13 20130621 prep Verd. P332 AgeI.Pst P334 AgeI.Pst P336 AgeI.PstI.png

TUM13 20130621 prep Verd. P338 AgeI.Pst P8 AgeI.XbaI.png

  • Extracted via QIAGEN gel extraction kit, digested PCR product purified via PCR Purification Kit


[Expand] 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)
  • Ligation batch for F116+F118 (TEV-Linker-TM-GFP+SERK-SigP_NLuc)


  • Ligation batch for F116+F119 (TEV-Linker-TM-GFP+SERK-SigP_SpyCatcher)
  • Ligation batch for F116+F120 (TEV-Linker-TM-GFP+SERK-SigP_SpyTag)


  • Ligation batch for F116+F121 (TEV-Linker-TM-GFP+SERK-SigP_XylE)


  • Ligation batch for F123+F124 (Laccase+pSB1C3)
  • Negative control was prepared by replacing the volume of the insert by ddH2O.
  • The ligation was performed for 1 hour at room temperature.


[Expand] 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.

[Expand] 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:

[Expand] 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:
  • The content has been mixed with a pipette
  • The PCR program was performed after following scheme:
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

[Expand] 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

[Expand] 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)

Reaction batch for P342 (AlcR after QCI):

PCR cycling parameters - P316 (npt-casette after QCI) and P342 (AlcR after QCI):

[Expand] 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:

[Expand] 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
  • 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


[Expand] 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)

Preparative digestion of P160 (SV40 NLS)


Preparative digestion of P249 (Thermonuclease)


Preparative digestion of PCR of IRES)

  • 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

  • Extracted via QIAGEN gel extraction kit, digested PCR product purified via PCR Purification Kit

[Expand] 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
  • Ligation batch for F125+F96 (N-TEV-linker in pSB1C3+PIF3)
  • Ligation batch for F124+F41 (IREs+pSB1C3)
  • Negative control was prepared by replacing the volume of the insert by ddH2O.
  • The ligation was performed for 1 hour at room temperature.


[Expand] 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.

[Expand] 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

[Expand] QuikChange II of pActin (P359, P360)

Investigator: Katrin

Aim of the experiment: QuikChangeII of pActin (P359, P360) Procedure:

Reaction batch

PCR cycling parameters

[Expand] 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)


[Expand] Analytical digestion and gelelectrophoresis of P347-P358

Investigator: Katrin

Aim of the experiment: Analytical digestion and gelelectrophoresis of P347-P358

Procedure:

  • 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


TUM13 20130623 anal Verd. P355-P358 EcoRI.PstI.png

[Expand] 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.

[Expand] 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

[Expand] 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:


Week 10

Monday, June 24th

[Expand] 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
  • Analytical digestion with EcoRI and PstI
  • 60 min incubation, 37 °C
  • Gel elektrophoresis on 1% agarose gel

TUM13 20130624 anal Gel P361-P366 EcoRI.PstI.png

[Expand] 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

[Expand] 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:

[Expand] 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.

[Expand] 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)
  • Ligation batch for F109 + F130 (SERK-SigP + Laccase)
  • Ligation batch for F113 + F131 (PhyB_36AA-Linker_C-TEV + IRES)
  • Ligation batch for F115 + F131 (TEV + IRES)
  • Negative control was prepared by replacing the volume of the insert by ddH2O.
  • The ligation was performed for 1 hour at room temperature.

[Expand] 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.

[Expand] 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:

  • 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

[Expand] 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

[Expand] 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:

[Expand] 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.

[Expand] 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:

[Expand] 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
  • Master-Mix for P374-P375 and control P316
  • Master-Mix for P376-P377 and control P342
  • Batch for analytical digestion of P367-P377 and controls
  • 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

TUM13 20130625 anal gel QCIInpt XbaI.PstI QCIIAlcR XbaI.SpeI.png

[Expand] 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
  • Batch for P35


  • 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


[Expand] 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

[Expand] 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:

[Expand] 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:

  • 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

[Expand] 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

[Expand] 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:


Thursday, June 27th

[Expand] 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:


[Expand] 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.

[Expand] 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:
  • The content has been mixed with a pipette
  • The PCR program was performed after following scheme:
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

[Expand] 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

[Expand] 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

  • 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

TUM13 20130627 anal Verd P380 P385 P309 P383 P386 P390 P392 XbaI.EcoRI.png


[Expand] 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)

  • 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

TUM13 20130627 präp Verd P361 XbaI.PstI P364 XbaI.PstI P266 EcoRI.AgeI.png

TUM13 20130627 präp Verd P388 AgeI.PstI P382 NgoMIV.PstI P247 EcorI.NgoMIV.png

TUM13 20130627 präp Verd P379 AgeI.PstI P374 EcoRI.XbaI P165 EcoI.SpeI.png

  • Extracted via QIAGEN gel extraction kit, digested PCR product purified via PCR Purification Kit

[Expand] 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):
  • Ligation batch for F136 + F137 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF6) (vector:insert = 1:3):
  • Ligation batch for F136 + F138 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF3) (vector:insert = 1:3):
  • Ligation batch for F143 + F144 (npt-csette + t35S) (vector:insert = 1:3):
  • Negative control was prepared by replacing the volume of the insert by ddH2O.
  • The ligation was performed overnight at 16 °C.

Friday, June 28th


[Expand] 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.


[Expand] QuikChange III of AlcR (P376)

Investigator: Andi, Johanna

Aim of the experiment: QuikChange III of AlcR (P376)

Reaction batch

PCR cycling parameters

[Expand] 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)

reaction batch for P246(Nanoluc)


reaction batch for F133(PCR of pActin)

  • 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


[Expand] 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)
  • Ligation batch for F145+F42 (pActin + pSB1C3) (batch: 30 ng vector!)


  • Negative controls were prepared by replacing the volume of the insert by ddH2O.
  • The ligation was performed for 1 hour at room temperature.

[Expand] 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



[Expand] 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


[Expand] 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.

[Expand] 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


[Expand] 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

[Expand] 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.

[Expand] 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

  • Reaction batches were incubated for 3 h at 37 °C.

[Expand] 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

  • Gelpurified products were extracted via QIAquick gel extraction kit, QIAGEN.

[Expand] 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):
  • Reaction batch with Q5 Mastermix for P344 (FluA triple mutant amplification):
  • The content has been mixed with a pipette
  • The PCR program was performed after following scheme for P143 (pActin amplification):
  • The PCR program was performed after following scheme for P344 (FluA triple mutant amplification)
  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

Sunday, June 30th

[Expand] 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


[Expand] 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:
  • 23x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:
  • 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


TUM13 20130630 analVerd P411 P412 P246 P413 P414 P391 EcoRI.PstI.png


TUM13 20130630 analVerd P415-P418 P385 EcoRI.PstI.png

[Expand] 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):


  • The content has been mixed with a pipette
  • The PCR program was performed after following scheme for P143 (pActin amplification):
  • 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

[Expand] 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

  • 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


TUM13 20130630 prepVerd P404 EcoRI.XbaI P395 NgoMIV.SpeI F154 XbaI.AgeI.png

  • Extracted via QIAGEN gel extraction kit, digested PCR product purified via PCR Purification Kit

[Expand] 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):
  • 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):
  • F123 + F161 (FluA triple mutant in pSB1C3):

Week 11

Monday, July 1st

[Expand] Everyday I'm swaffeling

Investigator: Le swaffleur passive

[Expand] 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

Batch for preparative digestion of P393 (Stress inducible Promoter)with EcoRI and SpeI"

  • 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

  • Extracted via QIAGEN gel extraction kit, digested PCR product purified via PCR Purification Kit

[Expand] 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)
  • 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.

[Expand] 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.

[Expand] 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

[Expand] 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.

[Expand] 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.


[Expand] 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)
  • 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)
  • The ligation was performed for 2 hours at room temperature; the ligation batch was transferred to 16 °C afterwads over night.

[Expand] 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.

[Expand] 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):


  • The content has been mixed with a pipette
  • The PCR program was performed after following scheme for P143 (pActin amplification):
  • 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:
  • The reaction was performed for 1 h at 37 °C. Afterwards the mixture was inactivated by heat for 10 min at 75 °C.

[Expand] 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

Reaction batch 2

PCR cycling parameters

  • 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.


[Expand] 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):
  • Batch for preparative digestion of P358 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1):
  • Batch for preparative digestion of P395 (SpyTag):
  • 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

  • Extracted via QIAquick gel extraction kit, QIAGEN.

[Expand] 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:
  • 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:
  • Ligation was performed at RT for 1 h.

[Expand] 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.

[Expand] 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:
  • Ligation was performed at 16 °C overnight.

[Expand] 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.

[Expand] 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

[Expand] 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.

[Expand] 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.

[Expand] 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.

[Expand] 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


TUM13 20130703 analVerd P424-P428 SbfI.SpeI P404 XbaI.PstI P433-P435,P367 EcoRI.PstI.png



Thursday, July 4th

[Expand] 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

Reaction batch 2

PCR cycling parameters for batch 1

PCR cycling parameters for batch 2

  • 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)

[Expand] 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:
  • Ligation was performed at 22 °C for 1h
  • negative control was prepaed with water instead of insert F166

[Expand] 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

[Expand] 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.

[Expand] 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
  • Batch for preparative digestion of P412
  • Batch for preparative digestion of P462
  • Batch for preparative digestion of P453
  • Incubation at 37 °C; 3 h, then put into freezer


[Expand] 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:
  • Ligation batch for F141+F139
  • Ligation was performed at 22 °C for 1h
  • negative control was prepaed with water instead of inserts F147 and F139

Friday, July 5th

[Expand] 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
  • Ligation batch for F170+F168


  • Ligation was performed at 22 °C for 1h
  • negative control was prepaed with water instead of inserts F169 and F168

[Expand] 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.

[Expand] 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.

[Expand] 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:


[Expand] 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:


Reaction batch 2:

PCR cycling parameters for batch 1

PCR cycling parameters for batch 2

  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

[Expand] QuikChange of pYes (P3)

Investigator: Rosario

Aim of the experiment: QuikChange of pYes (P3)

Reaction batch

PCR cycling parameters

  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

[Expand] 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

[Expand] 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

[Expand] 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:


Reaction batch 2:

  • The two batches were cycled seperately for the first 10 rounds.

PCR cycling parameters

  • 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:

  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

[Expand] 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

[Expand] 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.

[Expand] 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

[Expand] 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.

[Expand] 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:
  • 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


TUM13 20130707 anal verd P468-P474 EcoRI.PstI.png

[Expand] 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):

  • The content has been mixed with a pipette
  • The PCR program was performed after following scheme for P378(PP1 amplification):

Cycling parameters:

  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
  • The resulting product was labeled as F174.

[Expand] 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

[Expand] 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):

Reaction batch for P464 (IgKappa-SigP_NanoLuc_SpyCatcher):

Reaction batch for P466 (IgKappa-SigP_NanoLuc_SpyTag):

  • 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

  • DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.

[Expand] 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):

Reaction batch for F174 (Proteinphosphotase 1):

  • Reaction batches were incubated at 37 °C for 4 h.
  • After digestion, the product was purified with QIAquick PCR Purification Kit, Qiagen.

[Expand] 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:

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:

Ligation batch for F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), vector:insert = 1:3:

Ligation batch for F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant), vector:insert = 1:3:

Ligation batch for F123+F175 (pSB1C3 only + PP1), vector:insert = 1:3:

  • Ligation was performed at 22 °C for 1 h
  • Negative controls were also prepared with water instead of insert.

[Expand] 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.

[Expand] 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

[Expand] 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:
  • 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

  • all quickchanges were successfull.

[Expand] 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:


Reaction batch 2:

  • The two batches were cycled seperately for the first 10 rounds.

PCR cycling parameters

  • 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:

  • Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.


[Expand] 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:


[Expand] 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 :


  • 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

  • DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.

[Expand] 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:

F92+F180 (IgKappa in pSB1C3 + LMU GFP), vector:insert = 1:3:


  • Ligation was performed at 22 °C for 1 h
  • Negative controls were also prepared with water instead of insert.

[Expand] 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

[Expand] 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.

[Expand] 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:
  • 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

TUM13 20130709 anal Verd P422 P494-P499 EcoRI.PstI.png

[Expand] 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

[Expand] 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:


Reaction batch 2:

  • The two batches were cycled seperately for the first 25 rounds.

PCR cycling parameters

  • 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:

  • 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

[Expand] 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):

  • After digestion, linearized DNA was purified with QIAquick PCR purification kit, QIAGEN.

[Expand] 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):

Reaction batch for P488 (IgKappa-SigP_SpyCatcher_NanoLuc):

Reaction batch for P492 (Proteinphosphotase 1)):

  • 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

  • DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.

[Expand] 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:

Ligation batch for F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), vector:insert = 1:3:

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:

Ligation batch for F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1), vector:insert = 1:3:

  • Ligation was performed at 16 °C overnight.
  • Negative controls were also prepared with water instead of insert.

Thursday, July 11th

[Expand] 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.

[Expand] 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:

Friday, July 12th

[Expand] 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:

Reaction batch #2, with 5% DMSO and O109 as oligonucleotide:

Reaction batch #3, O108 as oligonucleotide:

Reaction batch #4, O109 as oligonucleotide:

  • 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:

PCR cycling parameters with 65 °C annealing temperature:

  • 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:

PCR cycling parameters with 65 °C annealing temperature:

  • 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.

[Expand] 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

[Expand] 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

[Expand] 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.

[Expand] 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:
  • 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

Sunday, July 14th

[Expand] 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):

Reaction batch for P477 (pAct):

  • 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

  • DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.

[Expand] Ligation of F185+F186 (pAct + MCS_t35S_npt)

Investigator: Jeff

Procedure:

Ligation batch for F185+F186 (pAct + MCS_t35S_npt), vector:insert = 1:3:


  • Ligation was performed at 22 °C for 1 h
  • Negative controls were also prepared with water instead of insert.

[Expand] 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):

Reaction batch for P431 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES):

Reaction batch for P509 ((GGGGS)x5-TEV-site-linker_NLS_NucA):

Reaction batch for P510 (SERK-SigP_PP1)):

  • 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

TUM13 20130714 prepgel P510 AgeI.PstI.png

  • DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.

[Expand] 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:

Ligation batch for F190+F135 (SERK-SigP_PP1 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), vector:insert = 1:3:

  • Ligation was performed at 16 °C overnight.
  • Negative controls were also prepared with water instead of insert.

Week 13

Monday, July 15th

[Expand] 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

[Expand] Preparative digestion and gelelectrophoresis of all effectors

Investigator: Jeff

Procedure:

Mastermix:

  • 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

TUM13 20130716 prepgel 2.jpg


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


TUM13 20130716 prepgel 4.jpg


TUM13 20130716 prepgel 5.jpg TUM13 20130716 prepgel 5b.jpg


  • DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.

[Expand] 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.

[Expand] Analytical digestion and gelelectrophoresis of P520 - P525

Investigator: Jeff

Procedure:

  • 7x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:
  • 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

Wednesday, July 17th

Thursday, July 18th

[Expand] 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:

  • 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

[Expand] 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):

  • The content has been mixed with a pipette
  • The PCR program was performed after following scheme:

Cycling parameters:

  • 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

[Expand] Arrival of the correct pActin 5 from Physcomitrella patens from Reski lab

This plasmid was named p541

Tuesday, July 23rd

Wednesday, July 24th

[Expand] 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):

  • The content has been mixed with a pipette
  • The PCR program was performed after following scheme:

Cycling parameters:

  • After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
  • The resulting product was labeled as F208.

[Expand] 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

[Expand] Preparative digestion of P208 (PCR of PppActin5)

Investigator: Jeff


Procedure:

Reaction batch for F208 (PCR of PppActin5):


  • Reaction batch was incubated at 37 °C for 2 h.
  • After digestion, the product was purified with QIAquick PCR Purification Kit, Qiagen.


[Expand] Ligation and transformation of F209 + F41 (PppActin5 in pSB1C3)

Investigator: Jeff


Procedure:

Ligation batch for F209 (PppActin5), vector:insert = 1:3:


  • 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

[Expand] 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

[Expand] 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.

[Expand] Analytical digestion and gelelectrophoresis of P542 – P547

Investigator: Jeff

Procedure:

  • 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

Unfortunately the correctness of the clones could not be seen in this experiment. Thus a second digestion was performed:


  • 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


[Expand] 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:


Saturday, July 27th

Sunday, July 28st

Week 15

Monday, July 29th

[Expand] QuikChange of P544 (PppActin5)

Investigator: Jeff

Procedure:

PCR cycling parameters with 55 °C annealing temperature:

  • 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

[Expand] 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):

  • 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

  • DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.
  • Resulting fragment was named as F210.

[Expand] 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:

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:

  • Ligation was performed at 16 °C overnight.
  • Negative controls were also prepared with water instead of insert.

Thursday, August 1st

[Expand] 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

[Expand] 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

[Expand] 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.

[Expand] 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

  • 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

TUM13 20130803 analgel P562-P565 EcoRI.PstI.png

Sunday, August 4th

Week 16

Monday, August 5th

[Expand] 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:

Reaction batch for P558, P560, P562, P565:

  • 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

TUM13 20130805 prepgel P562 EcoRI.PstI P565 EcoRI.PstI.png

  • DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.
  • Resulting fragment was named as F214 – F218.

[Expand] 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):

Reaction batch for PCR with Q5 Mastermixes for P560 (eCFP):

  • 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:

  • 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.

[Expand] 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

Tuesday, August 6th

[Expand] 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.

[Expand] 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:

Reaction batch for F213:

  • Reaction batches were incubated at 37 °C for 2 h.
  • Digested products were purified with QIAquick PCR Purification Kit, QIAGEN.

[Expand] 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:

Ligation batch for F123+F220 (pSB1C3 + eCFP), vector:insert = 1:3:

  • Ligation was performed at 16 °C overnight.


[Expand] 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



[Expand] Preparative digestion of P428 (mMCS-T35S-npt) and P554 (PppAct5-QCII)

Investigator: Jeff


Procedure:

Reaction batch for P428:

Reaction batch for P554:

  • Reaction batches were incubated at 37 °C for 2 h.

TUM13 20130806 prepgel PPPAct5diezwote.jpg

The bands were extracted from the gel and termed F221 and F222. The concentrations were determined to 42 and 37 ng/µl.


[Expand] 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:


  • Ligation was performed at room temperature overnight.


[Expand] 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

[Expand] 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.


[Expand] 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

[Expand] 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

All these clones were discarded.
Additionally the fragments for the ligation were controlled.

[Expand] 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

[Expand] 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.

[Expand] 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

  • 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

TUM13 20130809 analgel PppAct5mMCST35Snpt Gel2 EcoRI.jpg

TUM13 20130809 analgel PppAct5mMCST35Snpt Gel3 EcoRI.jpg

TUM13 20130809 analgel PppAct5mMCST35Snpt Gel4 EcoRI.jpg

Saturday, August 10th

[Expand] 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.

[Expand] 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

  • 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

Sunday, August 11th

[Expand] Preparative digestion of P615 (SYFP2 RFC25)

Investigator: Jeff

Aim of the experiment: Preparative digestion of P615 (SYFP2 RFC25)

Procedure:

[Expand] Preparative digestion and gelelectrophoresis of P617 (eCFP RFC25)

Investigator: Jeff

Aim of the experiment: Preparative digestion of P617 (eCFP RFC25)

Procedure:

[Expand] 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:

  • Ligation was performed at 16 °C overnight.

[Expand] 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

[Expand] 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:


[Expand] 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.

[Expand] 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

[Expand] 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
  • Ligation batch for self-ligation of F227


[Expand] 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.

[Expand] 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

[Expand] 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

[Expand] 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
  • batch for digestion of P615
  • batch for digestion of P637 and P639
  • 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

[Expand] 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:

[Expand] 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:

[Expand] 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:

[Expand] Preparative digestion of P644 (banana odour), P648 & P649 (PppAct5-mMCS-T35S-npt)

Investigator: Florian


Procedure:

Reaction batch for P644:

Reaction batch for P648:

Reaction batch for P649:

  • Reaction batches were incubated at 37 °C for 3 h.

TUM13 20130806 prepgel PPPAct5diezwote.jpg

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

[Expand] Preparative digestion of P414 and P640 + Preparative Gelelectrophoresis + Gelextraction

Investigator: Andi


Procedure:

Reaction batch for P414:


Reaction batch for P640:

  • 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

  • Gelextraction was performed after preparative Gelelectrophoresis
  • The resulting fragments were named F231 (Laccase) + F232

[Expand] 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


Ligation batch for F230+F192 (plasmid with mini MCS + catecholdioxygenase cytoplasmatic), vector:insert = 1:3

Ligation batch for F230+F193 (plasmid with mini MCS + EreB cytoplasmatic), vector:insert = 1:3

Ligation batch for F230+F194 (plasmid with mini MCS + Nanoluciferase cytoplasmatic), vector:insert = 1:3

Ligation batch for F230+F195 (plasmid with mini MCS + nanoluciferase secretory_SERK), vector:insert = 1:3

Ligation batch for F230+F196 (plasmid with mini MCS + gutathiontransferase cytoplasmatic), vector:insert = 1:3

Ligation batch for F230+F197 (plasmid with mini MCS + EreB membrane associated), vector:insert = 1:3

Ligation batch for F230+F198 (plasmid with mini MCS + Nanoluc membrane associated), vector:insert = 1:3


Ligation batch for F230+F199 (plasmid with mini MCS + nanoluc secretory_Ig Kappa), vector:insert = 1:3

Ligation batch for F230+F200 (plasmid with mini MCS + Nanoluc+IRES+Spytag_Nanoluc), vector:insert = 1:3

Ligation batch for F230+F201 (plasmid with mini MCS + Nanoluc+IRES+Nanoluc_Spycatcher), vector:insert = 1:3

Ligation batch for F230+F202 (plasmid with mini MCS + Nanoluc+IRES+Nanoluc_Spytag), vector:insert = 1:3

Ligation batch for F230+F203 (plasmid with mini MCS + IgKappa_GFP secretory), vector:insert = 1:3

Ligation batch for F230+F204 (plasmid with mini MCS + Nanoluc+IRES+Spycatcher_Nanoluc), vector:insert = 1:3


Ligation batch for F230+F205 (plasmid with mini MCS + FluA membrane associated), vector:insert = 1:3

Ligation batch for F230+F214 (plasmid with mini MCS + PP1 membrane associated), vector:insert = 1:3

Ligation batch for F230+F217 (plasmid with mini MCS + PIF3 safety), vector:insert = 1:3

Ligation batch for F230+F217 (plasmid with mini MCS + PIF3 safety), vector:insert = 3:1

Ligation batch for F230+F218 (plasmid with mini MCS + PIF6 safety), vector:insert = 1:3

Ligation batch for F230+F218 (plasmid with mini MCS + PIF3 safety), vector:insert = 3:1

  • Ligation was performed at roomtemperature, 1 h

[Expand] 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

  • Ligation was performed at room temperature, 1 h

[Expand] 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

[Expand] 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:

[Expand] 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:

  • Reaction batches were incubated at 37 °C for 3 h.

20130816 prepgel P640 EcoRI.NgoMIV.png

The band was extracted from the gel via QIAGEN Gel Extraction Kit and termed F233. The concentration was 31.1 ng/µl.


[Expand] 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.

[Expand] 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

  • neg. ctrl. w/ water instead of insert
  • Ligation was performed at room temperature, 1 h

[Expand] 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.

[Expand] 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


  • 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

[Expand] 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

[Expand] 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:

  • 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).

[Expand] 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

Ligation batch for F230+F235, vector:insert = 1:3

Ligation batch for F230+F236, vector:insert = 1:3

  • neg. ctrl. w/ water instead of insert
  • Ligation was performed at room temperature, 1 h

[Expand] 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:

[Expand] 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
  • 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

TUM13 20130818 anal verdau P699 bis P708 XbaI PstI.png

20130818 anal verdau P709 bis P712 XbaI PstI.jpg

[Expand] 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

[Expand] 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)

[Expand] Digestion of BITTE VERVOLLSTÄNDIGEN

Investigator: Ingmar

Aim of the experiment:

Procedure:

Week 18

Monday, August 19th

[Expand] 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

20130819 F245-F252.png

20130819 F253-F260.png

[Expand] 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.

Tuesday, August 20th

[Expand] 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:

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.

[Expand] 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:

[Expand] 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

  • neg. ctrl. w/ water instead of insert
  • Ligation was performed at room temperature, 1 h

[Expand] 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.

[Expand] 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

[Expand] 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


[Expand] 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

[Expand] 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.

[Expand] 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

  • 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


[Expand] 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.

[Expand] 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:

Reaction batch for P628,P629,P630, P632:

  • 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).

[Expand] 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
  • Ligation batch for F230+F263
  • Ligation batch for F230+F264, vector:insert = 1:3
  • Ligation batch for F230+F265, vector:insert = 1:3
  • Ligation batch for F230+F266, vector:insert = 1:3
  • Ligation batch for F230+F267, vector:insert = 1:3
  • neg. ctrl. water instead of insert.
  • Ligation was performed at room temperature for 1 h.

Friday, August 23rd

[Expand] Preparative digestion of P715 with XbaI and PstI + Preparative Gelelectrophoresis + Gelextraction

Investigator: Louise, Flo


Procedure:

Reaction batch for P715:

  • 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

[Expand] 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.

[Expand] 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:

[Expand] 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
  • Ligation batch for F188+F268, vector:insert = 1:3


  • neg. ctrl. water instead of insert
  • Ligation was performed at room temperature for 1 h


[Expand] 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.

[Expand] 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.

[Expand] 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

[Expand] 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.

[Expand] 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
  • Batch for 500ml of a 50 mM CaCl2-solution
  • Batch for 50ml of a 50 mM CaCl2-solution, 15% v/v Glycerin
  • Autoclave each bottle of solution and leave each in a 4°C room.


[Expand] 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.

[Expand] 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.

[Expand] 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
  • Ligation batch for F230+F263
  • Ligation batch for F230+F264, vector:insert = 1:3
  • Ligation batch for F230+F265, vector:insert = 1:3
  • Ligation batch for F230+F266, vector:insert = 1:3
  • Ligation batch for F230+F267, vector:insert = 1:3
  • neg. ctrl. water instead of insert.
  • Ligation was performed at 16 °C overnight.

[Expand] 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:


Sunday, August 25th

[Expand] 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

[Expand] Ligation of F230+F228

Investigator: Johanna

Aim of the experiment: Ligation of F230+F228

Procedure:

  • Ligation batch for F230+F228, vector:insert = 1:3
  • neg. ctrl. water instead of insert.
  • Ligation was performed at room temperature for 1 h.

[Expand] 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

20130825 analgel P754-P757 P759 P760 EcoRI.png

20130825 analgel P742-P747EcoRI.PstI.png

[Expand] 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.

[Expand] 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

  • 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

[Expand] 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:

  • 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

[Expand] 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

[Expand] 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
  • Ligation batch for F188+F268, vector:insert = 1:3
  • neg. ctrl. water instead of insert
  • Ligation was performed at 16 °C over night

[Expand] 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.


[Expand] 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.


[Expand] 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

[Expand] 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

[Expand] 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.

[Expand] 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
  • 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

[Expand] 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.

[Expand] 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

  • 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

TUM13 20130826 analgel P791-P797 XbaI.PstI.png


[Expand] 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

[Expand] 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.

[Expand] 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

[Expand] 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.

[Expand] 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.

[Expand] 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:

Reaction batch for P799:

  • 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

[Expand] 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

  • 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


[Expand] 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).

[Expand] 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

[Expand] 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

[Expand] 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

[Expand] 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

  • Incubation at 37 °C for 2.5 h.
  • Preparative gelelectrophoresis at 90 V for 30 min on 1% agarose gel.

[[File:|500px]]

[Expand] 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
  • Ligation batch for F230+F274, vector:insert = 1:3
  • Ligation batch for F230+F275, vector:insert = 1:3
  • Ligation batch for F276+F277
  • 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

[Expand] 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).

[Expand] 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:

[Expand] 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


  • 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


TUM13 20130828 analgel P823-P833 XbaI.PstI 2.png


TUM13 20130828 analgel P834-P840 XbaI.PstI.png

Thursday, August 29th

[Expand] Midiprep cultures from Miniprep cultures P815 and P835

Investigator: Jeff

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) and P835 (SERK-SigP_SpyTag_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_SpyCatcher_NanoLuc + Plasmid mit Mini MCS)

Procedure:

  • 50 ml LB medium w/ 50 µl chloramphenicol stock (1:1000)
  • 1 ml preculture each