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

Kaur Jaanson via methods%40net.bio.net (by kjaanson from gmail.com)
Mon Feb 11 04:35:48 EST 2008


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
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>
>



-- 
Kaur Jaanson



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