cloning of PCR products - errors?

Frank O. Fackelmayer Frank at Fackelmayer.de
Thu Jun 28 12:01:49 EST 2001



Sergio wrote:
> 
> "Frank O. Fackelmayer" wrote:
> > catalog (page 75), the error rate for Taq it 285x10-6, that of PolI is
> > 9x10-6. That is a ratio of around 30.
> 
> Only 265x10-6 ??... damn... i must be doing something wrong. We use to get
> around 1x10-3 mutations when using Taq... almost five times the supposed error
> rate.

First, these numbers are for optimized PCR reactions. MgCl2 and dNTP
concentrations seem to be critical.
Second, experimentally determining the error rate is not easy, resulting
in a quite some variance between the numbers with different test systems.

Irrespective of the actual numbers, I have stopped using Taq for
anything but analytical PCRs or the preparation of hybridization probes,
and use a proofreading enzyme (Pfu) for all amplifications of DNA that
will be cloned.


Frank




More information about the Methods mailing list