SSCP PAGE problems

RJ Wessell rwessell at magnus.acs.ohio-state.edu
Wed Oct 18 14:46:11 EST 1995


In article <Pine.A32.3.91.951017190736.120727A-100000 at spider.usp.br>,
fgdnobre at usp.br (Francisco G da Nobrega ib - bio 7588) wrote:

> On 16 Oct 1995, Ayo Ola wrote:
> 
> > I'm trying to screen some DNA samples for mutations using the SSCP=20
> > method.  After running my  5% PAGE + 10% glycerol gel for 15hrs at room=
> =20
> > temp my gels have refused to stick onto the Whatman paper and every=20
> > attempt to dry the gel ends up losing the gel. My gel is made up as ffws=
> =20
> >      16ml 50% glycerol
> >       8ml  50% Acrylamide Bis(49:1)
> >       4ml 10X TBE
> >       0.48ml 10% APS
> >       0.08ml TEMED
> >       51.44ml dH20
> > Running buffer used is 0.5% TBE.
> > I would be very grateful if anyone could help me out as I'm running=20
> > crazy.
> > Please mail replies, thank you.
> >=20
> > Ayo
> >=20
> Dear Ayo:
> 
> You could forget about drying and just remove one of the glass plates and=
> =20
> cover the gel with Saran. Put it inside on of the Kodak cardboard=20
> cassettes (or the Sigma flexible plastic ones) over your Xray film with a=
> =20
> intensifying screen underneath and expose OVN. That's how we usually do=20
> with the Maxam and Gilbert sequencing.=20
> 

   This is a very workable idea, but I would like to add another idea
which is currently working for me.  I run non-radioactive samples out,
then seperate the plates, place the plate with the gel stuck to it into a
staining jar and spread SYBR Green gel stain onto it.  After half an hour,
I pour off the stain (which can be reused several times) put Saran Wrap
over the gel, and lay it down on a UV box and expose it for photography. 
This is fairly easy and gives immediate results.  The drawback is the
expense of the SYBR Green.  If you do have the money, though, I would
recommend it.  It's available from FMC Bioproducts.

                                       R.J. Wessells



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