R: CHEF gel genomic DNA restriction(No virus check: scan engine not ready)

Sudheendra Rao N R via methods%40net.bio.net (by sudhee26 from gmail.com)
Mon Feb 11 23:43:57 EST 2008


Hey,
i found this one on net.
check if it can troubleshoot some of your problems.
I think looking again into your protocol would do lot of help.

http://humgen.wustl.edu/hdk_lab_manual/12/12_2.html

Sudheendra.

On Feb 11, 2008 3:05 PM, Kaur Jaanson <kjaanson from gmail.com> wrote:

> Hi
>
> Sorry, forgot to add the conditions.
>
> I use 1.2% agarose gel with 0.5x TAE buffer, running parameters are
> 5.4 V/cm field strength, 2-90 sec switch time, 19 hr run time.
> Biorad CHEF DR-II system to run the pulse field electrophoresis.
>
> Kaur Jaanson
>
> On Feb 11, 2008 11:23 AM, Prof. Piero Sestili <piero.sestili from uniurb.it>
> wrote:
> > Dear Kaur,
> >
> > post the running conditions you're using then we can talk about the CHEF
> > protocol modifications.
> >
> > Cheers
> >
> > Prof. Piero Sestili
> > Istituto di Farmacologia e Farmacognosia e
> > Centro di Ricerca sull'Attività Motoria
> > Università degli Studi di Urbino "Carlo Bo"
> > Via "I Maggetti" 26
> > 61029 URBINO (PU)
> > Tel. 0722 303414; 0722 305524
> > Fax 0722 303401
> >
> >
> > -----Messaggio originale-----
> > Da: methods-bounces from oat.bio.indiana.edu
> > [mailto:methods-bounces from oat.bio.indiana.edu] Per conto di
> > kjaanson from gmail.com
> > Inviato: domenica 10 febbraio 2008 21.57
> > A: methods from magpie.bio.indiana.edu
> > Oggetto: CHEF gel genomic DNA restriction(No virus check: scan engine
> > not ready)
> >
> > Hi
> >
> > I have lately tried to restrict the genomic DNA of mammalian cells in
> > agarose plugs and then separate the fragments with CHEF pulse field.
> > The restriction seems to be working but the restricted DNA migrates
> > weird in gel. It is very dense in the Kb-Mb range (EtBr staining shows
> > that the DNA in that range has kind of spread over the lane borders).
> > After the dense part ends the DNA occupies only the central part of
> > the lane. Southern plot of the gel shows that the DNA runs higher
> > (more slowly) than it should be, also the bands are a bit distorted.
> >
> > I was wondering if the anomaly might be associated with the
> > concentration of DNA in agarose plug, to my calculations there should
> > be about 3ug of genomic DNA in one plug. If that is the case then
> > could I vary the pulse field parameters so that the high amount of
> > genomic DNA would migrate normally?
> >
> >
> > Kaur Jaanson
> > _______________________________________________
> > Methods mailing list
> > Methods from net.bio.net
> > http://www.bio.net/biomail/listinfo/methods
> >
> >
>
>
>
> --
>  Kaur Jaanson
>
> _______________________________________________
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>



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