a***e
发帖数: 829 | 1 偶用的是two-step PCR, 把要突变的点设计在一对互补的引物上，最好在中间，第一步用
normal ５' primer+mutant 3'primer以及mutant 5'primer+normal 3'primer分别得到基
因前段和后段的PCR产物，第二步用正常的5' and 3' primers 做引物，以第一步PCR的两
个产物为模板，得到含有突变的全长DNA，再clone到质粒上拿去测序鉴定就行乐。 | a***o
发帖数: 129 | 2 not really, you have actually same # of steps as 2-step PCR
One step: PCR----DpnI-----Self ligation
two step: PCR----PCR---TA cloning
And actually your steps are more tricky, if you consider that Pfu has low
processivity while you have to PCR the full length of the plasmid which is at
least 3kb, you didn't really save time; and also compared to the efficiency
of 2-step which is 100%, the efficiency of your method is low; another
consideration may not be so important to you. you have to buy ex | a***o
发帖数: 129 | 3 Good point. OK, this is the best-kept secret of the century, hehe. The truth
is I always do my first round of PCR with Pfu (Pfx from invitrogen seems to be
quite efficient). If not for ease of cloning I will do second round with it
too. But since the fragment you want to mutate is usually around only 1 kb,
taq will be good enough.
Another reason I prefer Pfx in first round is that Taq should introduce an
overhang at the end of the product, sometimes this maks you troube in second
round by introd |
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