Stillman Lab writes:
> I am using a recombinant PCR technique to create transcriptional fusions
> between two homologous genes. I have recovered several clones which
> contain Taq induced mutations, and I am trying to find a better enzyme to
> perform my PCR reactions.
> Stratagene markets the Pfu Polymerase which is supposed to have a much
> lower error rate than either Taq or Vent. Has anyone actually used Pfu
> and compared the rate himself? If someone has used Pfu in PCR could you
> tell me if the extension time and/or temperature varies from those for
> Taq. Thanks in advance for your reply.
>>Helen_McBride at hlthsci.med.utah.edu
We have used Pfu for cloning experiments. After several
constructs and sequencing, we have found about 1 clone in 10
has a mutation (not bad I think). Generally, the sizes have
ranged from 300 - 1100 bases.
The Pfu salt conditions are different than Taq (10mM vs 50mM).
that corresponds to a reduction in the melting temperature by
about 10 - 11 degrees. We use the Lander Primer program which
allows input of salt concentration so it hasn't been a problem