ABI 373A DNA Sequencer data just fades away

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Mon Feb 13 13:54:23 EST 1995


Paul Morrison writes

[extensive description of a sporadic abberation affecting
sequence in some regions of an automated seq. gel]

My experience is limited to manual seq., but I am struck by the
similarity of your problem to the glycerol artifact.  That is,
the sporadic nature (related to the way people sporadically
decide more enzyme is better), and the position and nature of
the artifact (bands spread out but not missing, and only high
on the gel) sounds just like the glycerol effect.  I don't
know if your sample work up removes glycerol, but if you're
not using glycerol tolerant buffer, you might try it.  

More generally, it might be anything in the samples that varies
from sample to sample, runs as a high broad band on the gel, and
interacts with the DNA running along with it.  You say that the
same template will give the problem one day and not the next.  I
would suggest taking such a template and doing a series where the
amount of template used varies from way too much to less than usually
used.  If the problem reproducibly sets in on the high end of the 
template, then you're bringing something in with the template that's
causing the problem, and the sporadicity is related to how much
this substance gets concentrated on the way to the gel.  A similar
strategy aimed at other components (primers, enz) might finger the 
problem.  

Good luck; sounds like a tough problem.
Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at uthscsa.edu




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