WangLab:Deletion and Addition Inserts: Difference between revisions
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(New page: ===***=== 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/l): 0.625 # primre 2 (100ng/l)...) |
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| Line 3: | Line 3: | ||
# 10x buf: 2.5 | # 10x buf: 2.5 | ||
# DNA (miniprep): 1, 1(1/5), 1(1/10). | # DNA (miniprep): 1, 1(1/5), 1(1/10). | ||
# primer1 (100ng/ | # primer1 (100ng/ul): 0.625 | ||
# primre 2 (100ng/ | # primre 2 (100ng/u): 0.625 | ||
# H2O: 19.25 | # H2O: 19.25 | ||
# dNTP: 0.5 | # dNTP: 0.5 | ||
Latest revision as of 12:55, 23 May 2007
***
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
***
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
***
group 2 gives the best results.
