SSCP PAGE problems
rwessell at magnus.acs.ohio-state.edu
Wed Oct 18 14:46:11 EST 1995
In article <Pine.A184.108.40.2061017190736.120727A-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=
> > 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=
> > 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.
> > Ayo
> Dear Ayo:
> You could forget about drying and just remove one of the glass plates and=
> 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=
> 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.
More information about the Methods