WangLab:Deletion and Addition Inserts

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Deletion and Addition using Site-direct mutagenesis Kit from Strategene

  1. 10x buf: 2.5
  2. DNA (miniprep): 1, 1(1/5), 1(1/10).
  3. primer1 (100ng/ul): 0.625
  4. primre 2 (100ng/u): 0.625
  5. H2O: 19.25
  6. dNTP: 0.5
  7. pfu Turbo: 0.5

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mix 4 groups: 10xbuf, primer1, primer2, H2O: 2.5X4=10, 0.625X4=2.5, 0.625X4=2.5, 19.25X4=77, aliquote 2.5+0.625+0.625+19.25=23 to each tube. Add Dntp mix, DNA, pfu Turbo: 0.5, 1, 0.5

Run program Prset:

  1. cycle 1, 95oC, 30 sec
  2. cycle 18, 95oC, 30 sec
    • 55oC, 1min
    • 68oC, 12 min

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group 2 gives the best results.

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