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

[Kaur Jaanson]

Hi

I have already read that manual, unfortunately it doesn't say anything
about the DNA concentrations effect on the mobility.
I am currently trying to test if the restriction enzyme buffer might
cause the problem.

On Feb 12, 2008 6:43 AM, Sudheendra Rao N R <sudhee26 from gmail.com> wrote:
> 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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> > Methods from net.bio.net
> > http://www.bio.net/biomail/listinfo/methods
> >
>
>
>
> --
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> Think before you nod
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-- 
Kaur Jaanson