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. 

The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Dr. Duncan Clark
DNAmp Ltd.
TEl/FAX 01252376288

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