cloning of PCR products - errors?
Frank O. Fackelmayer
Frank at Fackelmayer.de
Thu Jun 28 12:01:49 EST 2001
> "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
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.
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