Vent PCR conditions

awalley at molbiol.ox.ac.uk awalley at molbiol.ox.ac.uk
Mon Nov 14 07:25:10 EST 1994


Hi,

I'm trying to switch from ordinary Taq polymerase to a proofreading enzyme in 
my PCR to reduce a high level of Taq errors I was getting in cloned PCR 
products. My PCR reaction is 45s at 94, 45s at 61, 20s at 72 for 35 cycles. 
Are any of these stages too long or is 35 cycles too many? I've reduced 
enzyme and primer concentrations but I still get three products at 500-1500bp 
rather than the 327bp product I'm expecting.

Help!

Andrew Walley,
IMM, Oxford, UK 



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