WangLab:Multiple Mutations

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Contents

Multiple mutations (using the stratagene multi kit):

  1. 10x buf: 2.5
  2. DNA(template, miniprep): 1, 1(1/2), 1(1/5), 1(1/10)
  3. primer1 (100ng/ul): 1
  4. primer2 (100ng/ul): 1
  5. primer3 (100ng/ul): 1
  6. dntp_mix: 1
  7. Enzyme: 1
  8. H2O: add until total 25 ul =16.5

Mix

Mix 10x buf, primer1,2,3, H2O together, for 5 groups: 2.5X5=12.5, 1X5=5, 1X5=5, 1X5=5, 16.5X5=82.5, aliquote 2.5+16.5+1+1+1=22 to each tube, add DNA, DNTP, Enzyme, 1, 1,1.

Run Prset_multi

  1. cycle 1, 95oC, 1 min
  2. cycle 30, 95oC, 1min
    • 55oC, 1min
    • 65oC, 12 min

group 4 gives the best results.

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