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Still got them mutagenesis blues!

Michael Szardenings msz at bio.embnet.se
Tue Dec 12 05:47:23 EST 1995

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.  

Dear Yasha,
don't worry, its quite normal to obtain at least 2 DNA species after the
primer extension/fill in (or whatsoever ppl call this)-reaction. I have
actually once seen more and did not have any problems with the
transformation. There should be at least small amount of ccc-DNA (some ppl
call covalently closed circular DNA) to yield some clones, if your ligase
is really working. If not, then you should indeed not abtain any clones in
Kunkel method, as the template will be gone before the internal ligase
manages to fix the nick. Have you tried this with a template containing T
only ('non-Kunkel')? Moreover, have you tried to repeat an old experiment,
perhaps you have a more specific problem with your present experiment?
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