joining two fragments with PCR

Dover Nir dovern at LEONARDO.LS.HUJI.AC.IL
Tue Oct 6 07:55:30 EST 1998


We are doing this routinely for site directed mutagenesis. A critical
point is that the Polymerase buffer should be fresh not one that have
been defrosted. What we usually do is to defrost the buffer once, divide
it, referees and use each tube once. Another point could be the joining
temperature I usually use much lower temp. then the Tm, about 50-55C. A
third point is the amount of DNA, use allot of DNA (up to 200-400 ng
each)in aquimolar ratio.
If you need more help write to me yoramg2 at leonardo.ls.huji.ac.il




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