Team:LMU-TUM Munich/Labjournal
Labjournal
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Week Test
Monday, April 22nd
Transformation of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)
Investigator: Jeff, Rosario
Aim of the experiment: Transformation of Phytochrome B for protein fusion.
Procedure:
- CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
- 2 µl of DNA was added to 100 µl of competent cells and gently mixed.
- 30 min incubation on ice
- 5 min. heat shock at 37 °C
- Adding of 1 ml LB-medium to each tube.
- Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
- 100 µl of the cell suspension was plated on one chloramphenicol plate.
- The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
- The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
Miniprep of pTUM100 with pGAL, pTEF1, pTEF2, pADH and RFC25 compatible RFP generator
Investigator: Jeff, Rosario
Aim of the experiment: Miniprep of pTUM100 with pGAL, pTEF1, pTEF2, pADH and RFC25 compatible RFP generator
Procedure:
- Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
Sequencing of RFP-Generator (RFC25, pSB1C3)
Investigator: Jeff, Rosario
Aim of the experiment: Sequencing of RFP-Generator (RFC25, pSB1C3)
Procedure:
Sequencing batch were prepared after manufacturer's protocol. (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer)
Tuesday, April 23rd
Picking of of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)
Investigator: Jeff, Rosario, Florian
Aim of the experiment: Picking of of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)
Procedure:
- pSB1C3 plasmid with BBa_K801031 (PhyB 2 - 908 aa, RFC25): Colonies were picked from chloramphenicol plates.
- Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
- 4 colonies were picked.
- These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight
Analytical digestion and gelelectrophoresis of RFP-generator (RFC25, pSB1C3, P4 & P5)
Investigator: Jeff, Rosario, Florian
Aim of the experiment: Analytical digestion and gelelectrophoresis of RFP-generator (RFC25, pSB1C3, P4 & P5).
Procedure:
- Batch for analytical digestion for P4 with NgoMIV+AgeI-HF
| volume | reagent |
| 2.5 µl | Plasmid DNA P4 |
| 2 µl | NEBuffer 4 (10x) |
| 0.25 µl | NgoMIV (10 U/µl) |
| 0.25 µl | AgeI-HF (20 U/µl) |
| 15 µl | ddH2O |
| =20 µl | TOTAL |
- Batch for analytical digestion for P5 with NgoMIV+AgeI-HF
| volume | reagent |
| 2.5 µl | Plasmid DNA P5 |
| 2 µl | NEBuffer 4 (10x) |
| 0.25 µl | NgoMIV (10 U/µl) |
| 0.25 µl | AgeI-HF (20 U/µl) |
| 15 µl | ddH2O |
| =20 µl | TOTAL |
- Incubation for 90 min at 37 °C.
- Analytical gelelectrophoresis was performed at 90 V for 60 min.
Results:
| 1 kbp ladder DNA ladder | P4 | P5 |
| Mutation successful | Mutation successful! |
- Parts are compliant and do not contain RFC25 forbidden restriction sites.
Sequencing of pTUM vectors with pGAL, pADH, pTEF1, pTEF2
Investigator: Jeff, Rosario, Florian
Aim of the experiment: Sequencing of pTUM vectors with pGAL, pADH, pTEF1, pTEF2
Procedure:
Sequencing batch were prepared after manufacturer's protocol. (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The different vectors we sequenced received the following barcodes:
- ADH in pTUM100: FR01002265
- TEF1 in pTUM100: FR01002266
- TEF2 in pTUM100: FR01002266
- GAL in pTUM100: FR01002268
Sequencing of TEF2 in pTUM100 was not interpretable. The other sequences were consistent with the sequences in the parts registry.
Wednesday, April 24th
Miniprep of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)
Investigator: Jeff, Florian
Aim of the experiment: Miniprep of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3).
Procedure:
- Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
Analytical digestion and gelelectrophoresis of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3), P7 - P10
Investigator: Jeff, Florian
Aim of the experiment: Analytical digestion and gelelectrophoresis of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3), P7 - P10.
Procedure:
- Batch for analytical digestion for P7 with NgoMIV+AgeI-HF
| volume | reagent |
| 2.5 µl | Plasmid DNA P7 |
| 2 µl | NEBuffer 4 (10x) |
| 0.25 µl | NgoMIV (10 U/µl) |
| 0.25 µl | AgeI-HF (20 U/µl) |
| 15 µl | ddH2O |
| =20 µl | TOTAL |
- Batch for analytical digestion for P8 with NgoMIV+AgeI-HF
| volume | reagent |
| 2.5 µl | Plasmid DNA P8 |
| 2 µl | NEBuffer 4 (10x) |
| 0.25 µl | NgoMIV (10 U/µl) |
| 0.25 µl | AgeI-HF (20 U/µl) |
| 15 µl | ddH2O |
| =20 µl | TOTAL |
- Batch for analytical digestion for P9 with NgoMIV+AgeI-HF
| volume | reagent |
| 2.5 µl | Plasmid DNA P9 |
| 2 µl | NEBuffer 4 (10x) |
| 0.25 µl | NgoMIV (10 U/µl) |
| 0.25 µl | AgeI-HF (20 U/µl) |
| 15 µl | ddH2O |
| =20 µl | TOTAL |
- Batch for analytical digestion for P10 with NgoMIV+AgeI-HF
| volume | reagent |
| 2.5 µl | Plasmid DNA P10 |
| 2 µl | NEBuffer 4 (10x) |
| 0.25 µl | NgoMIV (10 U/µl) |
| 0.25 µl | AgeI-HF (20 U/µl) |
| 15 µl | ddH2O |
| =20 µl | TOTAL |
- Incubation for 90 min at 37 °C.
- Analytical gelelectrophoresis was performed at 90 V for 60 min.
Results:
| 1 kbp ladder DNA ladder | P7 | P8 | P9 | P10 |
| Part is correct | Part is correct | Part is correct | Part is correct |
Transformation of E. coli XL1 blue with
Investigator:
Aim of the experiment: Transformation of 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.
- 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 µl LB-medium to each tube.
- The cell suspension was plated on ampicillin plates (inclusive rescue plate) and incubated over night at 37 °C in the cell-culture shaker.
Week 2
Thursday, June 23rd
Miniprep of E. coli Xl1-Blue transformed with ligation product P80/81 (mRuby3 K1/2), P82/83 (EspP K1/2), P84/85 (StrepTag K1/2) and Trafo of K157001
Investigator: Jan, Julian
Aim of the experiment: Miniprep of E. coli Xl1-Blue transformed with ligation product F50(K1,2), F51(K1,2), F52(K1,2) and Trafo of K157001 Procedure:
- Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
- Concentrations:
| Plasmid | c [ng/µl] |
| P80 | 432,7 |
| P81 | 294,8 |
| P82 | 450,5 |
| P83 | 479,0 |
| P84 | 108,0 |
| P85 | 356,0 |
| P86 | 47,2 |
Friday, June 24th
Analytical digestion and gelelectrophoresis of P83 (EspP K2) and P83 (StrepTag K2)
Investigator: Julian, Niklas, Luisa
Aim of the experiment: Analytical digestion and gelelectrophoresis of P82/83 (EspP K1/2) and P84/85 (StrepTag K1/2).
Procedure:
- Batch for analytical digestion for P82-P85 with EcoRI-HF
| volume | reagent |
| 0.5/1.0 µl | Plasmid DNA (-/P84) |
| 1 µl | CutSmart buffer (10x) |
| 0.5 µl | EcoRI-HF(10 U/µl) |
| 8/7.5 µl | ddH2O (-/P84) |
| =10 µl | TOTAL |
Sequencing of P80(mRuby3 K1), P83 (EspP K2) and P85 (StrepTag K2)
Investigator: Julian
Aim of the experiment: Sequencing of P80(mRuby3 K1), P83 (EspP K2) and P85 (StrepTag K2)
Procedure:
Sequencing batch were prepared after manufacturer's protocol. (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer). Sequencing primer VF2 was used
The different vectors we sequenced received the following barcodes:
- mRuby3 in pSB1C3 (P80): FR11326590
- EspP in pSB1C3 (P83): FR11326588
- Streptag in pSB1C3 (P85): FR11326587
Transformation of E. coli XL1 blue with F64 (quickchanged P3(pSAm1))
Investigator: Niklas
Aim of the experiment: Transformation of 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.
- 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 750 µl LB-medium to each tube.
- The cell suspension was plated on ampicillin plates (inclusive rescue plate) and incubated over night at 37 °C in the cell-culture shaker.
--> Quickchange did not work, do again, than new transformation!
Gelextraction of F67(BirA), F68(mRuby), F69(EGFR-TMD), F70(pSB1C3) and F71(pSB1C3)
Investigator: Niklas
Aim of the experiment: Gelextraction of F67(BirA(Digest. F59 [EcoRI; SpeI])), F68(mRuby(Digest. F60 [NgoMIV; SpeI])), F69(EGFR-TMD(Digest. F60 [NgoMIV; SpeI]), F70(pSB1C3(digest. P74 [NgoMIV; SpeI]) and F71(pSB1C3(digest. P74 [EcoRI; SpeI])
Procedure:
Gelextraction was performed by manufacturers protocol (Qiagen).
Saturday, June 25th
Miniprep of E. coli Xl1-Blue transformed with P60 (mRuby/EGFR),F58 (Ligation pASK75 + Streptactin), F65 (CMV + CD4), F66 (CMV + EGFR), P70 (Short Linker)
Investigator: Niklas
Procedure:
- Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
- Concentrations:
| Plasmid | c [ng/µl] |
| P87 | 81 |
| P88 | 34,5 |
| P89 | 86,3 |
| P90 | 108,5 |
| P91 | 417,4 |
Analytical digestion and gelelectrophoresis of F64 (quickchanged P3) for verification of succesful quickchange
Investigator: Niklas
Procedure:
- Analytical digestion with NdeI and gelelectrophoresis. If quickchange worked there should be a band at about 3200 bp (only one restriction site left)
- Incubation over night at room temperature.
- Analytical gelelectrophoresis was performed at 90 V for 60 min.
Results:
File:Muc16 Quickchange NA.JPG.JPG
Just a band showing a few bp (Primer), there is no plasmid band -> Quickchange did not work
Inoculation of colonies from Ligation of F69 + F70 (EGFR-TMD in pSB1C3)and F44 + F30 (mRuby in pSB1C3)
Investigator: Niklas
Procedure:
- 6x 4 ml LB+Cam media
- Each culture was inoculated with one colony
- Incubation at 37°C overnight
Sunday, June 26th
Miniprep of E. coli Xl1-Blue transformed with ligation product F44 + F30 (mRuby3 in pSB1C3), F66 + F70 (CMV+EGFR in pSB1C3)
Investigator: Luisa
Aim of the experiment: Extracting F44 + F30 (mRuby3 in pSB1C3), F66 + F70 (CMV+EGFR in pSB1C3) from E.coli XL-1-blue
Procedure:
- Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
- Concentrations:
| Plasmid | c [ng/µl] |
| P92 | 162,9 |
| P93 | 447,9 |
Repetition of Quick-Change PCR of P3 (pASK + SAm1)
Investigator: Luisa
Procedure:
- The QC-PCR was performed according the SOP.
- Reaction Mix:
| volume | reagent |
| 1,25 µl | Primer O21 |
| 1,25 µl | Primer O22 |
| 1 µl | dNTP-mix |
| 5 µl | Pfu-Ultra-II reaction buffer |
| 1 µl | template DNA (1:10 dilution of p3) |
| 0,5 µl | Pfu-Ultra-II Polymerase |
| 40,5 µl | ddH2O |
- digestion of PCR-Product with DpnI for 1h at 37°C
- now labeled P94
Transformation of E. coli XL1 blue with P94 (quickchanged P3(pSAm1))
Investigator: Luisa
Aim of the experiment: Transformation of 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.
- 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 950 µl LB-medium to each tube.
- The cell suspension was plated on ampicillin plates (inclusive rescue plate) for pASK (F72) and on chloramphenicol plates for P92 and P93 and incubated over night at 37 °C in the incubator.
Analytical digestion and gelelectrophoresis of P92 (mRuby), P93 (CMV + EGFR) and P94 (pASK75)
Investigator: Luisa
Aim of the experiment: Analytical digestion and gelelectrophoresis of P92 (mRuby), P93 (CMV + EGFR) and P94 (pASK75).
Procedure:
- Batch for analytical digestion for P92 and P93 with EcoRI-HF and PstI-HF
| volume | reagent |
| 1 µl | Plasmid DNA |
| 1 µl | CutSmart buffer (10x) |
| 0.5 µl | EcoRI-HF(10 U/µl) and PstI-HF (10 U/µl) for P92/93, NdeI (10 U/µl) for P94 |
| 7.5/7 µl | ddH2O |
| =10 µl | TOTAL |
Results:
- 1. band (P92): no mRuby at 700bp visible, only empty vector
- 2. band (P93): empty vector and EGFR --> perfect
- 3. band (P94): no signal at all --> repetition of QC-PCR
Week 3
Monday, June 27th
Sequencing of P67 (EGFR-Signalpeptid)
Investigator: Niklas
Procedure:
Sequencing batch was prepared after manufacturer's protocol. (15 µl of plasmid DNA (100 ng) and 2 µl sequencing primer (VF2))
FR11326586
Repetition of Quick-Change PCR of P3 (pASK + SAm1)
Investigator: Luisa
Procedure:
- The QC-PCR was performed according the SOP.
- Reaction Mix:
| volume | reagent |
| 1,25 µl | Primer O21 |
| 1,25 µl | Primer O22 |
| 1 µl | dNTP-mix |
| 5 µl | Pfu-Ultra-II reaction buffer |
| 1 µl | template DNA (1:10 dilution of p3) |
| 0,5 µl | Pfu-Ultra-II Polymerase |
| 40,5 µl | ddH2O |
- Digestion of PCR-Product with DpnI for 1h at 37°C.
- Transformation of 10µl into component E.coli XL-1-blue, according to SOP (1h incubation at 37°C necessary despite AmpR).
PCR of Genesynthesis 3 and 4
Investigator: Luisa
Aim of Experiment: Amplification of Genesynthesis 3 (contains BAP and IGKappa) and 4 (contains A3C5-tag and BM40)
Procedure:
- The PCR was performed according the SOP.
- Reaction Mix:
| volume | reagent |
| 2,5 µl | Primer VF2 |
| 2,5 µl | Primer VR2 |
| 1 µl | dNTP-mix |
| 10 µl | Q5 Polymerase reaction buffer |
| 1 µl | template DNA (1:10 dilution of p3) |
| 0,5 µl | Q5-Polymerase |
| 18 µl | ddH2O |
- Setup: iGEM_standard (Promega-cycler)
| temperature | time |
| 98°C | 2min |
| 98°C | 10sec |
| 66°C | 30sec |
| 72°C | 30sec |
| 72°C | 2min |
| 4°C | hold |
- the batches were then purified using the Quiagen PCR-Purification Kit.
Analytical digestion and gelelectrophoresis of P88 , P89 and P90
Investigator: Niklas
Aim of experiment: Analytical digestion and gelelectrophoresis of P88 (pASK75 + Streptactin, former F58), P89 (CMV + CD4, former F65) and P90 (CMV + EGFR-signal-peptide, former F66)
Procedure:
- Batches for analytical digestions:
P88: EcoRI
P89: EcoRI and PstI
P90: EcoRI
| volume | reagent |
| 5,8/2,3/1,8 µl | Plasmid DNA (P88/P89/P90) |
| 1 µl | CutSmart buffer (10x) |
| 0.5 µl | EcoRI-HF(10 U/µl)/ PstI |
| required amount for total volume of 10 µl | ddH2O |
Ligation of F67 and F71, Transformation of E. coli XL1 blue afterwards
Investigator: Niklas
Aim of the experiment: Ligation of F67 (BirA) and F71 (empty pSB1C3), Transformation of E. coli XL1 blue afterwards.
Procedure:
| volume | reagent |
| 2,4 µl | Vektor |
| 7,6 µl | Insert |
| 2 µl | 10X DNA-Ligase-buffer |
| 1 µl | T4-Ligase |
| 7 µl | ddH2O |
| =20 µl | TOTAL |
- CaCl2 competent E. coli XL1-Blue cells were taken out of stock in -80 °C freezer and were gently thawed on ice.
- 7 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 750 µl LB-medium to each tube.
- Incubation for 1 hour at 37 °C
- The cell suspension was plated on Cam-plates (inclusive rescue plate) and incubated over night at 37 °C in the cell-culture shaker.
- next step: analytic digestion of transformation was successful
Digestion of PCR on genesynthesis 3 and 4
Investigator: Luisa
Aim of experiment: Division of Leptin, IGKappa, A3C5, BM40 and BAP using SapI, HindIII, XbaI, AgeI for both batches.
Procedure:
- Batches for analytical digestions:
| volume | reagent |
| 1 µl each | enzyme (SapI, HindIII, XbaI, AgeI) |
| 5 µl | CutSmart buffer (10x) |
| 41 µl | DNA (purified PCR-products of GSY3 and 4) |
Ligation of F75 (mRuby) and F76 (NanoLuc) into F74 (pSB1C3 with Strep-Tag) and Transformation into E. coli XL1 blue
Investigator: Luisa
Aim of the experiment: Ligation of F75 (mRuby) and F76 (NanoLuc) into F74 (pSB1C3+Strep-Tag), Transformation of E. coli XL1 blue afterwards.
Procedure:
| volume | reagent |
| 4,3µl(for F75), 8,2µl (for F76) | Vector |
| 12,7µl (F75), 8,8 (F76) | Insert |
| 2 µl | 10X DNA-Ligase-buffer |
| 1 µl | T4-Ligase |
| =20 µl | TOTAL |
- Ligation was incubated at RT for 1,5h.
- CaCl2 competent E. coli XL1-Blue cells were taken out of stock in -80 °C freezer and were gently thawed on ice.
- 7 µl of DNA was added to 100 µl of competent cells and gently mixed.
- 15 min incubation on ice
- 5 min. heat shock at 37 °C
- Adding of 950 µl LB-medium to each tube.
- Incubation for 1 hour at 37 °C
- The cell suspension was plated on Cam-plates (inclusive rescue plate) and incubated over night at 37 °C in the incubator.
Chemical biotinylation of BSA
Investigator: Niklas
Procedure:
- BSA was chemically biotinylated with a 20x and 40x molar excess:
- 10 ml of 100 mM borate buffer with 50 mM NaCl (pH 8.85)
- dissolve BSA (10 mg/ml)
- Add biotin-NHS-ester: 20,5 mg for 40x molar excess
- reaction over night
