Team:Heidelberg/Templates/MM week8

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Contents

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:
Cycles temperature [°C] Time [s]
1 95 600
30 95 240
64 30
72 60
1 72 600
1 4 inf
Cycles temperature [°C] Time [s]
1 95 600
12 95 240
66°C ↓0.5°C 30
72 60
18 95 240
60°C 30
72 60
1 72 600
1 4 inf
  • 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
Cycles temperature [°C] Time [s]
1 98 10
5 98 1
45 5
72 130
25 98 1
55 5
72 120
1 72 120
1 4 inf
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
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