In article <msz-1212951147230001 at image.bmc.uu.se>, msz at bio.embnet.se
(Michael Szardenings) wrote:
> In article <Yasha-1112951213170001 at mac2wild2.tamu.edu>,
>Yasha at bioch.tamu.edu (Yasha Hartberg) wrote:
>> > I've posted this problem before but have a bit of new information to add
> > to the problem. I am using Kunkel selection site-directed mutagenesis
> > from an M13 derived template. We have used this method for years in the
> > lab with success. However, during the past year, I have not been able to
> > get plaques after transfecting with the extension reaction. Controls show
> > that my cells are competent and contain the F-factor. Direct
> > incorporation of P32-dATP in the extension shows that I am getting
> > extension. However, I always see two bands of equal intensity in the
> > autoradiogram of the extension reaction.
> > My first thought was that the two bands were the result of partial
> > linearization of the ssDNA template. Several template preps show the same
> > pattern, however, so I'm not sure.
We use Kunkel mutagenesis routinely, and the only problem we ever had was
using Promega T4 polymerase, which was contaminated with a uracil
glycosylase. I don't know if they've solved that problem, but you could
try changing your polymerase source.
University of Dundee