IUBio

loss of peak intensity at 2000 points

Robert Lyons boblyons at umich.edu
Sat Nov 22 07:18:10 EST 1997


Hamideh Zakeri wrote:
> 
> Once a while we experience partial loss of peak intensity at about 2000
> points while sequencing with ABI 377.  Sequences start very nicely,
> deminish around 2000-2500 points and come back very nicely afterward.  Any
> suggestion? Please write to me at hamideh at psts.wustl.edu.  Thanks a lot.
> 
> Hamideh
> 

This is a problem that has been reported by numerous labs in
various parts of the country. Our most common form was perfect
sequence out to ~200 nt, then fade for ~50-100 nt, then the
bands reappear, usually quite readable. Variations include:
smearing of the bands after the fade; fading *early* in the run;
no fade at all, but smearing starts right at the beginning
of the run. The problems have been worst on a 377, and to a
lesser extent on 373-Stretch and 373A (in that order).

I suspect these symptoms describe what Paul Morrison calls a
"fast fade gel". See his web site for additional info:

http://mbcf.dfci.harvard.edu/Docs/fastfade.html

   We'd eliminated just about every possible source - reagents,
gels, templates, primers etc etc etc. Eventually the plates
themselves were implicated. I know that does not make sense
given the symptoms, but sequencing is Voodoo, not science.
After many experiments in washing techniques, we tried using
MultiTerge (Clinic Allegiance Health Care, formerly Baxter
American Scientific). So far, in numerous tests, it has
worked. Plates that were rendered totally unusable have
been rescued and are again generating excellent sequence. Gels
pour better after the treatment and as a bonus, the bands appear
more intense. My 377 technician's current protocol is to do a
squirtbottle wash before *every* run, with 20% multi-terge.
   Here's the nitty-gritty: MultiTerge, VWR Scientific cat
#34171-010, 3.8L bottle. Soak the plates overnight in 5% Multi-
Terge, then wash as per your usual protocol before pouring a
gel. Alternatively, use 20% in a squirt bottle for frequent
washes. The detergent is not hazardous, and it's labeled as
being bio-degradable.
   Please pass on your experiences, either directly to me or
to this newsgroup.

Bob Lyons

-----------------------------
Robert Lyons, Ph.D.
Director, DNA Sequencing Core
University of Michigan
-----------------------------




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