Team:TU-Munich/Notebook/Labjournal
From 2013.igem.org
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* The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on chlorampenicol and ampicillin plates. | * The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on chlorampenicol and ampicillin plates. | ||
+ | </div> | ||
+ | |||
+ | <!--- this closes the week --> | ||
+ | </div> | ||
+ | |||
+ | =Week 1= | ||
+ | <div class="week" id="WWeek_1"> | ||
+ | =='''Monday, April 22nd'''== | ||
+ | |||
+ | <div class="general"> | ||
+ | === 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: | ||
+ | |||
+ | {|cellspacing="0" border="1" | ||
+ | |'''Plasmid''' | ||
+ | |'''c [ng/µl]''' | ||
+ | |- | ||
+ | |pRK792 | ||
+ | |214,5 | ||
+ | |- | ||
+ | |pRK793 | ||
+ | |134,3 | ||
+ | |- | ||
+ | |pRIL | ||
+ | |6,1 | ||
+ | |- | ||
+ | |ereA | ||
+ | |183,4 | ||
+ | |- | ||
+ | |ereB | ||
+ | |98,3 | ||
+ | |} | ||
+ | |||
+ | </div> | ||
+ | |||
+ | <div class="general"> | ||
+ | === 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) | ||
+ | |||
</div> | </div> | ||
Revision as of 15:24, 29 April 2013
Labjournal
Week 1
Monday, April 22nd
Transformation of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)
Investigator: Jeff, 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.
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)
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
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
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
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, 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).
Wednesday, April 24th
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)
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
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 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 1
Monday, April 22nd
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:
Plasmid | c [ng/µl] |
pRK792 | 214,5 |
pRK793 | 134,3 |
pRIL | 6,1 |
ereA | 183,4 |
ereB | 98,3 |
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)