Advice on ABI373A Sequencing Conditions?

Bruce Roe broe at aardvark.ucs.uoknor.edu
Mon Aug 1 08:34:00 EST 1994


In article <hardy-2907941515440001 at mighty.facs.fccc.edu>, hardy at mighty.fccc.edu (Richard R. Hardy) writes...
>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
Hi,
	Took us a while to figure out the optimal run conditions as well.
I'd suggest you stay with the 4.75% gels and try varying the Watts until
you get the spacing correct for the ABI software.  34W seems to be optimal
for our gels to give spacing in the 9-10 range.  Remember, spacing with a
value of -12 means it's not in the range needed by the ABI software.
	As for polymerization conditions, the stretch seems to be
more picky so settle on a protocol and stick to it all the time.

Cheers and good luck.....bruce
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