T4 DNA Polymerase-Buffer Conditions

Yasha Hartberg Yasha at bioch.tamu.edu
Wed Nov 2 03:07:26 EST 1994


I am revising our lab's protocol for site-directed mutagenesis.  Every
protocol I've read performs the synthesis reaction at pH 7.5.  However, the
quality control assays for T4 DNA polymerase activity are performed at pH
8.8.  I've talked with Clontech who said they had optimized the Transformer
Mutagenesis Kit at pH 7.5.  Promega told me that the optimum pH for T4
polymerase as a polymerase was pH 7.9.  In fact the technical support
person at Promega was amazed that the quality control was done at pH 8.8
and thought that it must have been a typo.  If it were a typo, then it is
industry-wide since Pharmacia also performs its quality control assays at
pH 8.8.

I would appreciate any insights anyone could provide into what would be the
optimal pH for mutagenisis, what is the optimum pH for T4 polymerase
activity, or why quality control assays might be done at non-optimal
conditions, or in fact why unit definitions might be at non-optimal
conditions.

Thanks.

Yasha Hartberg
Texas A&M University
"If people want to spend their whole lives creaming and tweezing and
brushing and tilting and gluing, that's really okay too, because it gives
them something to do."  Andy Warhol



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