Preparative PAGE for single stranded DNA?

bugg at mbcf.stjude.org bugg at mbcf.stjude.org
Tue Jun 9 18:03:33 EST 1992


In article <1421.2a2a4634 at mbcl.rutgers.edu>, hey at mbcl.rutgers.edu writes:
> I would like to try SSCP (single strand conformational polymorphism)
> analsis of heterozygous PCR products.  This will involve running
> an a preparative acrlymaide gel, staining w/ ethidum bromide, hopefully
> detecting single stranded products, and then isolating the bands of ]
> interest.  Does anyone have any insights on whether this will work.
> How much single stranded DNA must I have to see a band with
> ethidium in an acrylamide gel?  Can I get the DNA out of the gel? How?
> Thanks for any info.
> Jody Hey
> Rutgers University   hey at mbcl.rutgers.edu

I am not sure about the answers to all of your questions, but in my experience,
SSCP only works with a very low concentration of PCR product.  This is because
you are trying to prevent "snap-back" re-annealing of the denatured strands. 
It seems like the more DNA you use, the more double-stranded product you have
on your gel - which defeats the purpose of SSCP.   However, some people do SSCP
with single strand PCR products - I haven't tried this, but it would circumvent
the problem.  Let us know what you find out! 
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Barbara Bugg, M.S.  bugg at mbcf.stjude.org       "I refuse to have a battle of
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Memphis,  TN  USA
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