Team:TU-Munich/Notebook/Labjournal

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Week 1 22.4-28.4
Week 2 29.4-05.5
Week 3 06.5-12.5
Week 4 13.5-19.5
Week 5 20.5-26.5
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Labjournal

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, Leonie, 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, Leonie, 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, Leonie, 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, Leonie, 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, Leonie, 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, Leonie, 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, Leonie, 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, Leonie, 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, Leonie, 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 29nd

[Expand] Miniprep of pRK792, pRK793, pRIL, ereA, ereB

Investigator: Leonie

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:



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, Leonie, 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)



Investigator: Andi


[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: Leonie


[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,Leonie,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, Leonie

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: Leonie,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, Leonie

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, Leonie

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, Leonie, 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: Leonie, 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,Leonie,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, Leonie

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, Leonie

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, Leonie

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, Leonie

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, Leonie, Johanna, Jeff

Aim of the experiment: Analytical digestion and analytical gelelectrophoresis of ligation products P28, P117, P118, P119, P120, P121, P122, P123.

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:

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

Investigator: Jeff, Andi, Johanna, Rosario, Leonie

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, Leonie, 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 25st

[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, Leonie, 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) were successful .

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.


[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 were successful

Procedure:

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

[Expand] Analytical digestion and gelelectrophoresis

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.

Monday, May 27th

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

Investigator: Leonie,Johanna

Aim of the experiment: Oligohybridization of MiniMCS (MiniMCS fw (O56) and MiniMCS rev (O57) and of Spytag (Spytag fw (O54) and Spytag rev (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] Preparative digestion 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.
  • Incubation for 120 min at 37 °C.
  • Digested products were purified with QIAquick PCR Purification Kit, QIAGEN.
  • The digested fragments were labelled ASLDFKAÖSDF


  • Batch for preparative digestion of P39 (Middle-Linker) with PstI and AgeI.
  • Incubation for 120 min at 37 °C.
  • Digested products were purified with QIAquick PCR Purification Kit, QIAGEN.
  • The digested fragments were labelled KLASDÖFKAÖLSDFASD


  • Batch for preparative digestion of P20 (GFP-Psb1C3) with NgoMIV and PstI.
  • Incubation for 120 min at 37 °C.
  • Digested products were purified with QIAquick PCR Purification Kit, QIAGEN.
  • The digested fragments were labelled ASLDÖFKASÖLDFKASDF

[Expand] Quick Change mutagenesis to remove forbidden restriction sites - SDMIII - of P125(Laccase), P126 (EreB)

Investigator: Volker, Andi

Aim of the experiment: Removal of forbidden restiction sites from P125 (Laccase), P126 (EreB).

Procedure: Quickchange - PCR

Reaction batch - P125

Reaction batch - P126

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.