SSCP help

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Wed Sep 7 17:47:37 EST 1994


Frederick Garbrecht writes:

> I am trying to do a single stranded conformational polymorphism 
> analysis on a ~500 bp PCR fragment.  I run the formamide denatured 
> fragments out alongside non-denatured control fragments on a 4% PA 
> gel and then silver stain.  The non-denatured fragments give nice 
> sharp bands, but the denatured fragments give a fairly broad smear, 
> in which I cannot discern individual bands.

We had the same problem.  Our SSCP bands were not that sharp to start
with.  This was compounded by the PCR fragment of this size assuming
multiple conformations (different hairpinning options I presume) and
hence making multiple bands.  The result was an indistinct pattern
which was also not real reproducible.  The best I was able to do was
to go to shorter PCR products.  Also, of course, end labelling one
primer reduces the complexity of the pattern by half.  Maybe someone
else will know a way to improve the gel itself.

Steve Hardies, Dept. of Biochemistry, Univ. of Texas HSC at San Antonio
Hardies at thorin.uthscsa.edu




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