Team:Heidelberg/Templates/MM week8

From 2013.igem.org

2013-06-17

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG. Lanes 1-6: annealing at 66°C; lanes 8-11: touch-down 66->60°C; lane 6: NEB 2-log; lane 1,7: negative control (BAP1-pLF03); lane 2,8: colony-PCR of colony electroporated with gel-extracted PCR product; rest: colony-PCRs of colonies electroporated with PCR-purified PCR product.

• repeat colony PCR, use 1 µl of liquid culture from gel-extracted plate, pick colonies from PCR-purified plate (Taq, 25 µl total volume)
• 2 conditions:

• no positives
• plate samples of liquid cultures from 2013-06-15 on Amp, grow at 37°C

2013-06-18

• colonies grew on Amp

2013-06-19

Gel electrophoresis of the PCR amplificate. Lane 1: NEB 2-log ladder; lanes 2-5: PCR with Phusion Flash (2 µl of product were put on gel)

• prepare for repetition of experiment: run 4 PCRs of 1 ng pLF03 (miniPrep from 2013-06-04) with primers ygfG21C1 and ygfG21C2 using Phusion Flash (performed by Dominik), one reaction = 50 µl total volume

Gel electrophoresis of the purified PCR amplificate. Lane 1: NEB 2-log ladder; lane 2: purified PCR with Phusion Flash (1 µl of product were put on gel)

• pool products, purify (digestion only 3 h)
• nanoDrop: 2088 ng/µl -> remeasure: 532 ng/µl
• load 1 µl purified PCR product on gel -> very weak band -> nanoDrop wrong
• to eliminate error: make fresh electrocompetent cells:
  • pick colony from BAP1-pKD46 plate from 2013-06-04
  • inoculate 1 ml LB + Amp
  • grow at 30°C

2013-06-20

• too little cells: use aliquot from 2013-06-06
• use all available DNA (14 µl) and 100 µl cells (complete aliquot)
• grow in SOC + IPTG(1mM) at 300 rpm for 3h
• spin cells down, decant supernatant, resuspend in remainder of medium
• plate 10 µl, rest on Cm + IPTG