D1S80 Ladder-help?

Garry P. Larson glarson at ccmail.llu.edu
Tue Jan 23 20:43:55 EST 1996


I have been trying to amplify the ABI D1S80 allelic ladder with little
success.  I've diluted the stock ladder 2,000X and tried it directly.  My
goal is to amplify the D1S80 ladder with F-dUTP's and run it on a ABI 373
using the Genescan mode.  What appears to be a simple task-amplifying the
ladder- has become an obstacle.  Is there some inhibitor present in the
ladder, though it shouldn't be a problem at a 1/2,000X dilution?  The stock
ladder has each allele present at app. 4ng/ul.  I realize its not necessary
to use the ladder since one can size the fragments internally land by lane
but I'd like it for student demonstration purposes.

btw, if you could reccommend a specific gel setup (acrylamide vs. long
ranger, native vs. denaturing) to separate the alleles (app. 400-950bp)
that too would be useful.  ABI has thus far been unable to reccommend a
setup.

The primers and PCR conditions are those that are published by numerous
sources, so that's most likely not the problem.  Any enlightening comments
would be greatly appreciated.  If you also know of a URL that lists PCR
primers AND their rxn. conditions this too would be useful (I found a human
genome related site once that listed such a thing - and of course forgot to
bookmark it!)

Thanks in advance,
GLarson



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