Which strand of a plasmid is replicated?

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Thu Sep 14 10:53:51 EST 2000


This message has been posted by:  612 right <i.hinners at REMOVE-THIS-TO-SENDicrf.icnet.uk>

????
No, after DpnI digest you would end up with a hybrid (some strands
repaired, some not) of nonmethylated DNA, after transformation both
replicate, each strand serving as a template
then you end up with some clones fine and others not... your eficiency
would go down and you will have to screen a lot.
I think its easier, cheaper and better to make 2 primers for the
mutagenesis.

??? or did I miss here something??
Regards, Ina



Wolfgang Schechinger wrote:

> Tim, thanks a big lot for this precise answer. May I conclude that it
> is theoretically sufficient to modify one strand (the lower strand in
> common sequence notations) for mutagenesis (by the quick change
> protocol) and to use a standard primer for the other one (in order to
> generate a fully non-methylated DNA necessary for the DpnI digest
> wich degenerates the template DNA)?
>
> All the best,
>
> Wolfgang
>
> > From:          tfitzwater at gilead.com
> > Subject:       Re:  Which strand of a plasmid is replicated?
> > Date:          13 Sep 2000 20:40:38 +0100
> > Organization:  BIOSCI/MRC Human Genome Mapping Project Resource Centre
> > X-To:          methods at hgmp.mrc.ac.uk
> > To:            methods at hgmp.mrc.ac.uk
>
> > >From: Wolfgang Schechinger
> > >(Wolfgang.Schechinger at med.uni-tuebingen.de) Date: Tue 12 Sep 2000 -
> > >20:45:12 BST
> >
> > >Hi all,
> > >Does anyone know which strand of a plasmid is replicated in E. coli
> > >K12 strains (I currently use 298)?
> >
> > >All input is welcome,
> >
> > >Wolfgang
> > >Dr. Wolfgang Schechinger, Dept. of Pathobiochemistry
> > >University of Tuebingen, Germany
> > >email: wolfgang.schechinger at med.uni-tuebingen.de
> > >wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
> >
> > Leading strand synthesis of ColE 1-type plasmids is directed by the
> > Primer RNA, which begins with the sequence 5' GCAAACAAAAAAACC... and
> > ends 555 nucleotides later with the sequence ...GGGGGGCGGACCUAUGGAAA
> > 3'.  DNA replication starts from RNase H cuts made at the final 3 A
> > residues and continues around the plasmid.  Lagging strand
> > replication starts at about position 720 downstream from the start
> > of Primer RNA transcription and is halted at about position 525 on
> > the non-template strand until the replication fork passes that
> > position.  Lagging strand synthesis then continues until two
> > concatenated double-stranded plasmids result.  These are then
> > resolved into separate entities.
> >
> > Tim Fitzwater
> > Principal Research Associate
> > Gilead Sciences
> >
> >
> > ---
> >
> >
> -----
> This message is encrypted. Use your brain to decode it.
> -----
> Dr. Wolfgang Schechinger, Dept. of Pathobiochemistry
> University of Tuebingen, Germany
> email: wolfgang.schechinger at med.uni-tuebingen.de
> wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
> -----
> usual disclaimers apply
> -----
>
> ---

--
Ina Hinners
ICRF
Secretory Pathways Laboratory
44 Lincolns Inn Fields
WC2A 3PX London
UK
email: I.Hinners at icrf.icnet.uk







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