PCR errors

seppen at amc.uva.nl seppen at amc.uva.nl
Wed May 31 07:17:25 EST 1995


In Article <3qf7n2$jnb at kadri.ut.ee>
mremm at tamm.eenet.ee (Maido Remm) writes:
>Hi,
>
>I just want to mention that I have always sequenced my inserts cloned by PCR
>and have NEVER met a mutation within amplified area (could be over 10 000 bp
>by now). I have used Taq from Fermentas, LITHUANIA and from Amersham.
>But I have found several mutations (around 10) within primer area, so primer
>synthesis seems to be more error-prone step.
>
>Maido Remm, 
>Estonian Biocentre and Tartu University  
>mremm at ebc.ee

well... here is my experience;
I cloned a 1 kb PCR fragment. The average was 2 mutations in
the whole fragment. When i sequenced different clones i found different
mutations. The taq i used was from promega, this is a recombinant taq. 
I have also tried taq in which the exonuclease activity was still present, with
low nucleotide concentrations and less cycles with the same results. Polymerase
with proofreading activity (VENT) did not work.
People in the lab next door got more or less the same results, different
fragment, different PCR machine.
It could be our water............
Greetings, 

Jurgen Seppen

Amsterdam
The Netherlands





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