3700 in core facility

mpento mpento at cmgm.stanford.edu
Tue Oct 24 11:19:39 EST 2000


What I try and have most customers do is have their DNA in water.  Some
DNA preps will use a buffer for the final elution.  Try and get them to
use water instead.  Do not use percipitation for your sequencing
reaction cleanup.  We use the millipore filterplate with sephadex G50.
Works well.  Be wary of sephadex plates that are already hydradrated
with buffer (best to have a wash step for these).  Be prepared for some
samples that do not run well on capillaries.  We get some poor
resolution samples that work well on a gel.  Sometimes diluting the
sample helps but it does not appear to be too much DNA?  Have you tried
running your "top heavy" samples a second time.  If the profile improves
than you know it was too many small ions in the sample.  If it stays the
same then that is the profile generated by your sequencing reaction.
You might try reducing your injection voltage and/or increasing your
injection time to improve the profile.  We use water as our loading
solution.  It works well and we do not see the failures that ABI say is
a potential problem.  We put our plate in just before (5-10min) the
sample transfer to the block and take it out after loading.  Also try
and get a good handle on the quality of your capillaries.  The range in
samples can be hard on them and you will need to know when they need to
be regenrated or replaced and wheather poor data is due to poor sample
or the capillaries starting to loose resolution.  Hope this helps  Good
luck
Martin








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