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Team:Heidelberg/Templates/MM week13
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2013-07-22
File:BAP1-colony-PCR2013-07-22.png
Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG (marked 2). Lane 1: NEB 2-log; lanes 2,4: BAP1-pLF03 (control); lanes 3,5: colony-PCR. Lanes 2,3: primers IK01+IK03; lanes 4,5: primers RB43+RB44
File:BAP1-colony-PCR2013-07-22 2.png
Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG (marked 2) with primers RB43+RB44. Lane 1: NEB 2-log; lane 2: BAP1-pLF03 (control); lane 3: colony-PCR; lane 4: streaked BAP1 by Konrad
- no positive PCR of colony yet: run colony-PCR with primers IK01+IK03 (iTaq, 20 ml total volume):
| Cycles | temperature [°C] | Time [s] |
|---|---|---|
| 1 | 95 | 300 |
| 12 | 95 | 60 |
| 68 ↓0.5°C | 30 | |
| 72 | 180 | |
| 23 | 95 | 60 |
| 62 | 30 | |
| 72 | 180 | |
| 1 | 72 | 600 |
| 1 | 12 | inf |
- primers RB43+RB44 (against sfp) iTaq, 20 µl total volume:
| Cycles | temperature [°C] | Time [s] |
|---|---|---|
| 1 | 95 | 300 |
| 35 | 95 | 60 |
| 55 | 30 | |
| 72 | 180 | |
| 1 | 12 | inf |
- unspecific bands in colony, no sfp bands at all
- re-run sfp PCR (primers RB43+RB44), include streaked BAP1 by Konrad
- sfp present in BAP1, not present in BAP1-pLF03, colony
- makes no sense, Konrad streaked his cells from the same batch of competent cells
- transfer BAP1, BAP1-pLF03, colony to liquid culture LB, grow at 37°C
2013-07-23
File:BAP1-colony-PCR2013-07-23.png
Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG (marked 2) and grown in liquid culture with primers RB43+RB44. Lane 1: NEB 2-log; lane 2: BAP1-pLF03 (control); lane 3: colony-PCR; lane 4: streaked BAP1 by Konrad, lane 5: BAP1-pMM64-pMM65 from 2013-06-11
- run colony-PCR with primers RB43+RB44 (iTaq, 20 µl total volume, use 1 µl of liquid culture)
| Cycles | temperature [°C] | Time [s] |
|---|---|---|
| 1 | 95 | 300 |
| 35 | 95 | 60 |
| 55 | 30 | |
| 72 | 180 | |
| 1 | 72 | 600 |
| 1 | 12 | inf |
- no sfp present
