PCR-SSCP ON ABI 373 WITH GENESCAN 672
buzardg at MAIL.NCIFCRF.GOV
Wed Mar 6 14:35:22 EST 1996
Four possibilities come to mind immediately. 1.) the bands are
there, but you have not run the gel long enough to see them, some fragments
can run anomalously slow; 2.) The gel has changed temperature dramatically
at some point, and the fragments were unable to make up there minds as to
which conformation was best, and in confusion just fuzzed-out; 3.) You have
the rare and extreme misfortune of having a fragment that both strands are
fuzzed out under the conditions used, you didn't do anything wrong per se;
4.) the concentration of product that you have after centriconing is so high
that re annealing has occurred, leaving only trace amounts of single strand
which you are failing to detect. This can be exasperated if you had an
unintentional slightly asymmetric PCR product, and the preponderant band is
a fuzz-out under your conditions. And there is the "Fred Schaffer" artifact,
a PCR product with a Clonetech p53 primer set that would not stay denatured
in my gel system, the strands re annealed in the gel at the interface as
they left the denaturing environment, again a rare foo-bah.
Try making an intentional asymmetric product with each of your
primers so that there is no possibility of re annealing. Run these products
side by side with your denature dsDNA and your undenatured product. Your
ssDNAs should run somewhere relative to a 400-800 bp dsDNA MWM, which you
must have on your gel also.
Try lowering your concentration of product.
Try using methyl mercury hydroxide instead of NaOH and Formamide.
Try changing the position of your primers by 10 bases in either
E-mail if you need more suggestions.
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