ecec at quads.uchicago.edu
Thu Oct 6 08:27:07 EST 1994
You ask about SSCP analysis. I wonder why you ask it here
rather than in the methods newgroup-to which I am redirecting
The basic SSCP procedure is pretty simple. The way I do it
(See Genetics 137:175-188) is as follows:
-Perform your PCR with a little bit of 32P labelled nucleotide included.
You can cut your PCR reaction volume down to 5 or 10 ul since you
probably won't need much product.
-Heat 1-3 ul of your PCR product together with 9 ul of denaturing
buffer at 94 C for 2-3 min. (denat. buffer is 95% formamide, 10mM NaOH,
0.5% bromphenol blue, 0.5% xylene cyanol; this is equivalent to
the left over stop solution from an otherwise exhausted sequencing kit).
-Chill on ice immediately for at least 5 minutes prior to electrophoresis
of the entire preparation on a polyacrylamide gel under non-denaturing
condditions. Don't let your gel get to hot! If you want to run it
quickly, then run it in a cold-room. You sometimes have to play around
a bit with the concentration of acrylamide and the degree of cross-linking
but straight out of the bottle gels for proteins should work fine for
a first approximation.
-Dry the gel onto a piece of 3mm chromatography paper and autoradiograph it.
-Instead of using 32P you can simply silver stain the gel. I originally
used silver staining myself, but found that others in our lab tended to
overexpose their gels (irreversibly), apparently out of "greed".
-If your PCR product is rather long, you might want to cut 5-8 ul with
an appropriate restriction enzyme in a 10 ul reaction volume,
and then use 1-3 ul of that in the denaturation step.
Needless to say, but I'll say it anyway, make sure to include both positive
and negative controls. If you have some alleles that you know differ by
a single base pair, then try them out first. It is also instructive to
run undenatured vs. denatured samples on the gel until you get used to
the technique-which shouldn't take long.
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