WangLab:Deletion and Addition Inserts
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Deletion and Addition using Site-direct mutagenesis Kit from Strategene
- 10x buf: 2.5
- DNA (miniprep): 1, 1(1/5), 1(1/10).
- primer1 (100ng/ul): 0.625
- primre 2 (100ng/u): 0.625
- H2O: 19.25
- dNTP: 0.5
- 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:
- cycle 1, 95oC, 30 sec
- cycle 18, 95oC, 30 sec
- 55oC, 1min
- 68oC, 12 min
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group 2 gives the best results.