Trudy,
Many, many people have been wrestling with a similar problem, with
varying degrees of success. The symptoms usually include smearing of
the bands (sometimes starting from the beginning, sometimes 200 nt
into the run), and may display a distinct fading just before the smear
begins. Our most common form was perfect sequence out to ~200 nt, then
fade for ~50-100 nt, then the lanes reappear, smeared and unreadable.
Through more experiments than I can count, we'd eliminated just
about every possible source of problems - reagents, gels, templates,
primers etc etc etc. Eventually the plates themselves were implicated.
I know that does not make sense given the symptoms, but hey, it's not
science, it's Voodoo. Numerous experiments tested various washing
protocols (Alconox variants, acids, bases) with unimpressive results.
Recently, our Field Applications Specialist passed on a note he
had received from another FAS suggesting the use of a commercial
detergent called MultiTerge (Clinic Allegiance Health Care, formerly
Baxter American Scientific). So far, in four separate tests, it has
worked like a charm! Plates that were rendered totally unusable have
been rescued and are again generating excellent sequence. Gels pour
better after the treatment and the bands are more intense besides. We
are continuing the tests to learn more about the failure and its cure,
but we are confident enough in the wash procedure to commit client
samples to it tonight.
Here's the nitty-gritty: MultiTerge, VWR Scientific cat #34171-010,
3.8L bottle. Soak the plates overnight in 5% MultiTerge, then wash as
per your usual protocol before pouring a gel. The detergent is not
hazardous, and it's labeled as being bio-degradable.
Please let me know if this cleans up your problem. I've CC'd my note
to Rob Brankamp, our FAS. I'm sure other ABI customers would like to
know if this works.
Bob Lyons
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Robert Lyons, Ph.D.
Director, DNA Sequencing Core
University of Michigan
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