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Team:Heidelberg/Templates/MM week8
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Contents |
2013-06-17
File:BAP1-colony-PCR2013-06-17.png
Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG. Lanes 1-6: annealing at 66°C; lanes 8-11: touch-down 66->60°C; lane 6: NEB 2-log; lane 1,7: negative control (BAP1-pLF03); lane 2,8: colony-PCR of colony electroporated with gel-extracted PCR product; rest: colony-PCRs of colonies electroporated with PCR-purified PCR product.
- repeat colony PCR, use 1 µl of liquid culture from gel-extracted plate, pick colonies from PCR-purified plate (Taq, 25 µl total volume)
- 2 conditions:
| Cycles | temperature [°C] | Time [s] |
|---|---|---|
| 1 | 95 | 600 |
| 30 | 95 | 240 |
| 64 | 30 | |
| 72 | 60 | |
| 1 | 72 | 600 |
| 1 | 4 | inf |
| Cycles | temperature [°C] | Time [s] |
|---|---|---|
| 1 | 95 | 600 |
| 12 | 95 | 240 |
| 66°C ↓0.5°C | 30 | |
| 72 | 60 | |
| 18 | 95 | 240 |
| 60°C | 30 | |
| 72 | 60 | |
| 1 | 72 | 600 |
| 1 | 4 | inf |
- no positives
- plate samples of liquid cultures from 2013-06-15 on Amp, grow at 37°C
2013-06-18
- colonies grew on Amp
2013-06-19
File:PLF03 PCR2013-06-19.png
Gel electrophoresis of the PCR amplificate. Lane 1: NEB 2-log ladder; lanes 2-5: PCR with Phusion Flash (2 µl of product were put on gel)
- prepare for repetition of experiment: run 4 PCRs of 1 ng pLF03 (miniPrep from 2013-06-04) with primers ygfG21C1 and ygfG21C2 using Phusion Flash (performed by Dominik), one reaction = 50 µl total volume
| Cycles | temperature [°C] | Time [s] |
|---|---|---|
| 1 | 98 | 10 |
| 5 | 98 | 1 |
| 45 | 5 | |
| 72 | 130 | |
| 25 | 98 | 1 |
| 55 | 5 | |
| 72 | 120 | |
| 1 | 72 | 120 |
| 1 | 4 | inf |
File:PLF03 PCR2013-06-19 purified.png
Gel electrophoresis of the purified PCR amplificate. Lane 1: NEB 2-log ladder; lane 2: purified PCR with Phusion Flash (1 µl of product were put on gel)
- pool products, purify (digestion only 3 h)
- nanoDrop: 2088 ng/µl -> remeasure: 532 ng/µl
- load 1 µl purified PCR product on gel -> very weak band -> nanoDrop wrong
- to eliminate error: make fresh electrocompetent cells:
- pick colony from BAP1-pKD46 plate from 2013-06-04
- inoculate 1 ml LB + Amp
- grow at 30°C
2013-06-20
- too little cells: use aliquot from 2013-06-06
- use all available DNA (14 µl) and 100 µl cells (complete aliquot)
- grow in SOC + IPTG(1mM) at 300 rpm for 3h
- spin cells down, decant supernatant, resuspend in remainder of medium
- plate 10 µl, rest on Cm + IPTG
