Simple question that is hard to answer

[peter]

On Feb 17, 3:34 am, "Peter Ellis" <p... from cam.ac.uk> wrote:
> peter wrote:
> > 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?
>
> >> Peter
>
> > Making heteroduplex on purpose...
>
> Why?  What's the goal of your experiments?  Why is it important to you to
> have both alleles present in one bacterium, and why can't you simply
> transform first with one allele and then the other?  Because that's the only
> way I can think of reliably getting both into the one cell, and even then
> it's hit and miss as to whether you'll retain both.
>
> It doesn't look to me as though cloning heteroduplex DNA is going to get you
> where you want to go.  It may be that in a tiny minority of cells you don't
> repair the mismatch, and end up heteroplasmic for the two alleles.  Or, if
> you just transform the mixture using a large excess of plasmid, a minority
> of cells will pick up more than one plasmid, and of that minority a small
> proportion will end up with both alleles.
>
> In each case, while you may get a very few colonies that carry both plasmids
> (but with no guarantee of preserving them both), you'll have a massive
> background level of colonies that only carry one or other of the alleles -
> and there won't be ably way or telling which is which, short of genotyping a
> gazillion colonies.
>
> Peter

Can't tell sorry....