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Advice on ABI373A Sequencing Conditions?

Richard R. Hardy hardy at mighty.fccc.edu
Fri Jul 29 15:15:44 EST 1994

We've just obtained an ABI373A-Stretch automated sequencer and have had
some problems getting good runs with the taq standard.  Using the 34cm
plates run with recommended 4.75% gels, the standards to define the matrix
ran OK, but the taq ran with many incompletely resolved peaks (lots of "N"
calls on the sequence analysis), clearly substandard.  Any
comments/suggestions as to what we should try?  Some have suggested that
the 34cm/4.75% combination doesn't give as good results as the old shorter
plate/6% gel combo and that electrophoresis conditions have to be varied
for optimization (we ran at 30W, some suggested 32).  Or perhaps the
polymerization conditions are more critical than with the old setup?  Any
help appreciated!

R. Hardy
Member, Institute for Cancer Research,
Fox Chase Cancer Center, Philadelphia, PA
(215) 728-2463

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