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Team:Heidelberg/Templates/MM week15
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Revision as of 16:52, 3 October 2013 by Nils.kurzawa (Talk | contribs)
2013-08-05
- run colony-PCR against sfp on BAP1-pKD46 with primers RB43+RB44 (iTaq, 20 µl total volume):
File:BAP1-colony-PCR2013-08-05.png
Colony-PCR of BAP1-pKD46 with primers RB43+RB44. Lane 1: sfp, lane 2: NEB 2-log
| Cycles | temperature [°C] | Time [s] |
|---|---|---|
| 1 | 95 | 300 |
| 35 | 95 | 60 |
| 55 | 30 | |
| 72 | 180 | |
| 1 | 72 | 600 |
| 1 | 10 | inf |
- inoculate 4 ml 2xYT+Amp with BAP1-pKD46, grow at 30°C
2013-08-06
File:Methylmalonyl-PCR2013-08-06 2.png
Lane 1: NEB 2-log; lane 3: colony-PCR with primers RB43+RB44
- inoculate 50 ml 2xYT+Amp+Ara(0.5%) with BAP1-pKD46, grow at 30°C until OD=0.71
- run colony-PCR with primers RB43+RB44 from 50 ml culture:
| Cycles | temperature [°C] | Time [s] |
|---|---|---|
| 1 | 95 | 300 |
| 35 | 95 | 60 |
| 55 | 30 | |
| 72 | 180 | |
| 1 | 72 | 600 |
| 1 | 10 | inf |
- prepare electrocompetent BAP1-pKD46
2013-08-08
- electroporate BAP1-pKD46 with 500 ng DNA (5 µl of PCR amplificate from 2013-07-08)
- grow in SOC + IPTG(1mM) + Arabinose (0.5%) at 37°C, 400 rpm for 3h
- spin cells down (3 min at 8000 rcf)
- plate 10 µl, rest on Cm, grow at 37°C
