stacking for DNA in PAGE??

Bruce Ritchings britchings at ucsd.edu
Thu Sep 11 18:01:40 EST 1997


Dear Netters,
	Here's an interesting technical question: when running SDS-PAGE gels of
proteins, we usually use a stacking gel (per Laemmli) to make the proteins run
as tight bands. Has anybody ever tried this when running DNA on non-denaturing
PAGE gels? My application is this: I'm trying to use the new "Storm"
phosphorimager (Molecular Dynamics, Inc.) in it's fluorescence mode to
precisely quantitate PCR products by running the PCR products on a 6%
non-denaturing acrylamide gel (regular 1% TBE buffer) and then post-staining
with a new dsDNA dye called Vistra Green. The dye works well, but my bands are
not sufficiently tight to see (and accurately quantitate) the faint bands I'm
looking for. If I could tighten up the bands I do see, I'm sure my approach
would work. Does anybody know how to get tighter dsDNA bands on a
non-denaturing PAGE gel? I thought of increasing the % of the gel, and that
might help a little, but my products are 550 bp, so if I increase the % of the
gel too much, the PCR products won't properly enter the gel (at least I think
they won't). My other thought was to use a stacking gel as one does with
proteins, but I'm afraid I'd have to try dozens of pH and gel% combinations to
get one that works. If anybody's figured this out already, I'd be appreciative
of your advice. Thanks, Bruce



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