Bp-exchange by PCR

Dr. Duncan Clark duncan at genesys.demon.co.uk
Tue Dec 10 11:45:42 EST 1996


In article <32AD2B0D.3E5D at urz.uni-heidelberg.de>, Klaus Lun
<klaus.lun at urz.uni-heidelberg.de> writes
>I have amplified a 1.2 kb fragment with Pfu Polymerase and after
>sequencing I found 2 Bp-exchanges, leading to  aminoacid substitutions.
>So I was wondering if this is the 'normal' mutation rate you get with
>Pfu-Polymerase; because I want  to express this fragment I absolutely
>need no Bp-exchange. So how many PCR-poducts should I clone ? 10, 20 ? I
>perform the PCR with 30 cycles ..
>So please, help me ..........
>
>       KLAUS

Fewer cycles mean fewer errors but of course less product. If you are
starting from Xsomal that could be a problem. If it is from a plasmid
clone then use a lot more template. 

Are you sure that the product you sequenced is incorrect? Could the
original sequence which you are referring to be the one that is wrong. 

Duncan  
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Dr. Duncan Clark
DNAmp Ltd.
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