Haynes Lab:Notebook/CRISPR Editing/2014/07/09

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Had about the same number of colonies on each of the ligation plates as the backbone. Will try the ligation following the Zhang lab protocol.

Will run through the protocol with pX330 and three gRNAs, g5, g13, g14

Annealing the oligos

oligo 1 (100μM) 1
oligo 2 (100μM) 1
10X T4 Ligation Buffer (NEB) 1
ddH2O 6.5
T4 PNK (NEB) 0.5

  • 37°C 30 min
  • 95°C 5 min
  • ramp down to 25°C at 5°C/min

Ligation reaction

pX330 (185ng/ul) 0.540540541
1:250 annealed oligos 2
10X Fast Digest buffer 2
10mM DTT 1
10mM ATP 1
BbsI 1
T7 DNA ligase 0.5
water 11.95945946


  • 37°C 5 min
  • 23°C 5 min
    • Cycle the previous two steps for 6 cycles (total run time 1h)
  • 4°C hold until ready to proceed

Long transformation, 75ul cells