Team:Heidelberg/Templates/MM week13

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2013-07-22

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
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

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
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