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Team:Heidelberg/Templates/MM week15
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Nils.kurzawa (Talk | contribs) m (Created page with "== 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|100px|thumb|right|Colony-PCR ...") |
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== 2013-08-05 == | == 2013-08-05 == | ||
* run colony-PCR against sfp on BAP1-pKD46 with primers RB43+RB44 (iTaq, 20 µl total volume): | * run colony-PCR against sfp on BAP1-pKD46 with primers RB43+RB44 (iTaq, 20 µl total volume): | ||
| - | [[File: | + | [[File:Heidelberg_BAP1-colony-PCR2013-08-05.png|100px|thumb| Colony-PCR of BAP1-pKD46 with primers RB43+RB44. Lane 1: sfp, lane 2: NEB 2-log]] |
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== 2013-08-06 == | == 2013-08-06 == | ||
| - | [[File: | + | [[File:Heidelberg_Methylmalonyl-PCR2013-08-06_2.png|100px|thumb| 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 | * 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: | * run colony-PCR with primers RB43+RB44 from 50 ml culture: | ||
Latest revision as of 02:36, 5 October 2013
2013-08-05
- run colony-PCR against sfp on BAP1-pKD46 with primers RB43+RB44 (iTaq, 20 µl total volume):
| 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
- 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
