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R: CHEF gel genomic DNA restriction(No virus check: scan engine notready)

R: CHEF gel genomic DNA restriction(No virus check: scan engine notready) - Protocols and Methods Forum

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Old 02-11-2008, 09:23 AM
Prof. Piero Sestili
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Default R: CHEF gel genomic DNA restriction(No virus check: scan engine notready)



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


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Inviato: domenica 10 febbraio 2008 21.57
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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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