Simple question that is hard to answer

Peter Ellis via methods%40net.bio.net (by pjie2 from cam.ac.uk)
Fri Feb 16 18:45:31 EST 2007


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?

Peter 




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