Simple question that is hard to answer

peter via methods%40net.bio.net (by peter.ianakiev from gmail.com)
Mon Feb 19 08:53:04 EST 2007


On Feb 19, 6:52 am, Duncan Clark <blackh... from abuse.plus.com> wrote:
> Historians believe that in newspost
> <1171648654.333893.138... from m58g2000cwm.googlegroups.com> on Fri, 16 Feb
> 2007, peter <peter.ianak... from gmail.com> penned the following literary
> masterpiece:
>
>
>
> >On Feb 16, 11:34 am, Duncan Clark <blackh... from abuse.plus.com> wrote:
> >> Historians believe that in newspost
> >> <a59bt2dap5kc69sqqjd0cd00f9p01pv... from 4ax.com> on Fri, 16 Feb 2007, ChenHA
> >> <h... from freeuk.com> penned the following literary masterpiece:
>
> >> >The Duet series are plasmids with
> >> >different replicons, so are different as far as the original question
> >> >is concerned.  The question would be one about plasmids with the same
> >> >replicon, and that is problematic.  If there are different antibiotics
> >> >used, then it may be possible to maintain the plasmids, but do
> >> >remember that antibiotics like ampicillin are broken down fairly
> >> >quickly, and when the antibiotics are gone, there is in fact no
> >> >selection, and one of the plasmids will most probably be lost.
>
> >> I was always curious about the replicons used in some clinical E.coli's
> >> that carry umpteen different plasmids. I remember many many years ago
> >> that one such E.coli was used for plasmid markers and had may 10
> >> different plasmids in it. They didn't appear to lose those plasmids
> >> despite no selection. May have been published in "Plasmid" back in the
> >> late 70's or early 80's.
>
> >> Duncan
> >> --
> >> I love deadlines. I especially like the whooshing noise they make as
> >> they go flying by.
>
> >> Duncan Clark
> >> GeneSys Ltd.
>
> >Duncan,
> >Your input is always appreciated, just my question was different and I
> >don't know how better to describe  - can one have two alleles of the
> >SAME Plasmid in the SAME cell, not different ori, not different
> >antibiotic resistances, everything is the same, just point muataion
> >that comes from cloning of heteroduplex DNA that has a mutation.
> >Peter
>
> Well a long long time ago before PCR, one made mutants using M13 ssDNA,
> annealed a mutagenic primer and extended around the M13 with Klenow, T7
> DNA pol or T4 DNA pol. Alternatively one used pEMBL vectors which were
> pUC18 plus an M13 F1 ori.
>
> I never heard of anyone getting out a heteroduplex at the end. If they
> had they would have got both mutant and wt protein expressed, which
> never happened.
>
> So I don't think you will be able to maintain a heteroduplex plasmid.
>
> I think you would get one homozygote plasmid per E.coli cell.
>
> There must be papers from years ago confirming or denying this, surely?
>
> Duncan
> --
> I love deadlines. I especially like the whooshing noise they make as
> they go flying by.
>
> Duncan Clark
> GeneSys Ltd.

Didn't they use uridilated DNA for templates? or that came later?
Anyway, I will make experiment and will find out. Thanks everyone for
the discussion.
Peter



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