Team:Heidelberg/Templates/MM week15

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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

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