Simple question that is hard to answer
(by peter.ianakiev from gmail.com)
Fri Feb 16 19:37:53 EST 2007
On Feb 16, 6:45 pm, "Peter Ellis" <p... from cam.ac.uk> wrote:
> peter wrote:
> > Peter,
> > Mismatch will be corrected indeed, but using what template - it seems
> > that it will be corrected again 50/50 and still end up as mixed
> > population?
> Doesn't matter what template. When you do the transformation, you'll only
> have *ONE* copy of the plasmid in each bacterium. Then, when it copies that
> for the first time, it'll correct using one strand or the other, and you'll
> have two identical copies of one allele or the other.
> > If that is the case and I isolate DNA and re-transform, I
> > should be able to get 50/50 of homozygous plasmids in the cells and
> > have colonies that have one or the other allele.
> Um, yes. If you have a mixed population and you transform it, some of the
> colonies will bear one allele and some will bear the other. What you will
> *not* find are single colonies that carry both alleles, or single bacteria
> that carry both alleles, or bacteria carrying uncorrected heteroduplexes.
> I'm not quite certain why you expect to have heteroduplex DNA in your
> transformation mixture in any case: how are you generating your plasmid for
> the transformation?
Making heteroduplex on purpose...
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