SSCP analysis/Help

Eric Cabot 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
follow-ups. 

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.


Alternates:
  -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.

-Eric Cabot
 

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