In article <hardy-2907941515440001 at mighty.facs.fccc.edu>,
hardy at mighty.fccc.edu (Richard R. Hardy) wrote:
> 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!
>after we received the stretch upgrade to our 373A, i also encountered
problems with the 4.75% gels as reccomended by ABI. our tech services rep,
when i told about this said that at that time, ABI had not finished testing
conditions for the stretch liner. i ran side by side gels using both 4.75%
and 6% gels and got infinitely better results with the 6% gel. we also run
at 32W instead of 30W which was set by the installer. i have actually been
getting better results using this combination (no other changes have been
made in our protocols) than will the 24cm well-to-read plates. the longer
running distance allows for greater separation at the laser-read region
which leads to more accurate sequencing.
also, do you clean up your template and primers prior to sequencing? we
EtOH precipitate our templates which are prepared using Wizard mini or maxi
preps, and also EtOH ppt. the primers we get from our in house core
facility. if we don't do this, we usually get very poor results as well.
hope this is of some help.
NOT BIG PANTS!!!
eric c. anderson
anderson at pharmdec.wustl.edu
660 s. euclid box 8103
washington univ. school of medicine
dept of molecular biol. and pharmacology
st. loser, misery 63110