Blotting DNA from polyacrylamide gels

LoPony ctfrench at wwisp.com
Tue May 30 23:22:51 EST 2000


Is there any way you could run a control gel for staining with EtBr and
photography and skip the staining of the gel used for the transfer?  EtBr
will inhibit transfer...

The UV nicking of the DNA before transfer if stained with ethidium is also a
reasonable approach to try.

Todd

--
==============================
Christopher Todd French
UAB Dept. of Comparative Medicine
1670 Univ. Blvd, VH 422
1530 3rd Ave. S
Birmingham, AL 35294-0019

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"Chris LaRosa" <clarosa at biocomp.unl.edu> wrote in message
news:392BF8FC.D56F4AAB at biocomp.unl.edu...
>
>
> Audrey Ah Fong wrote:
> >
> > Hi ! I'm having probelms regarding transferring DNA from polyacrylamide
> > gels.  I'm actually running DNA on DGGE gels (polaycrylamide with urea
and
> > formamide) and have to transfer DNA on nylon membranes.  The protocol I
> > have, used electroblotting but after staining the gels, the DNA,
especially
> > the bigger fragments are still there.  I do not know what i could be
doing
> > wrong.  To tell more precisely what I'm doing, after running the gel I
> > depurinate the gel for 5 minutes in 0.25 M HCL, rinsed in water followed
by
> > 5 minutes in 0.5 M NaOH.  I do neutralise for 5 min in 0.5 M Tris-HCl
and
>
>
> You can skip the depurination step simply by exposing your ETBR gel to
> the
> UV transilluminator for a couple of minutes.  In fact, some people in
> this building simply take a photograph.. the exposure seems to be enough
> to nick and cut the dna sufficiently to allow transfer.   I am not sure
> if this is critical to your problem. Your going to have to try or get
> advise on another doing exactly what your doing.







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