________________________________________________________________________
Robert B. Chadwick
Director, Genotyping/Sequencing Unit
Division of Human Cancer Genetics
Ohio State University
420 West 12th Avenue
Columbus, OH 43210
phone (614) 688-4783
FAX (614) 688-4761
e-mail chadwick-1@medctr.osu.edu
________________________________________________________________________
Bernd Weisshaar wrote:
> Problem description: there is a complete loss of signals in the lower
> region of the gel, and a "smeer" of green signal after that. In the upper
> part of the gel, the signals resume. Abi custom support only suggested
> better cleaning of glas plates, but the problem does not correlate with
> glas plate treatment. In one case, only half of the gel was affected (the
> gel was loaded in 4 steps, all probes were processed in parallel). If the
> samples are reloaded on a "good" gel, all is fine. But we have used the
> same chemicals and stocks for all gels .... where is the logic?
>Bernd -
>This is a problem that has plagued many sequencing labs.
>At one point, I scanned old emails and web sites and counted
>over two dozen locations reporting similar problems - including
>lots of the big sites, and ABI themselves. You will find that
>ABI is remarkably *un*helpful when trying to fix this. They
>really don't know the cause, nor can they suggest a reliable
>cure. I am quite disgusted by their lack of support and effort.
>The symptoms you report - blank areas of a gel and smeared
>data - are typical observations. You may also see (i) the blank
>region of the gel, followed by normal sequence, (ii) the smear
>region but no blank region, and (iii) any combination of these
>followed by a gradual restoration of normal data. You also
>pointed out that these symptoms may arise in some areas of a gel
>yet not others. You may see them associated with standards as
>well as "real" samples.
>While it may not seem logical, the greatest contributing
>factor really does seem to be the glass plates. There is weak
>evidence that the samples contribute to the failure, and that
>the specific gel mix you use can help or hurt. However, we can
>reliably resolve the smear/fade problem by switching to new
>glass plates. Obviously this is a short-term solution, since we
>can't spend $500 on plates every time we see a smear.
>It appears that there is some chemical buildup on sequencing
>plates. ABI does not know what it is. Several suggestions have
>been put forth by sequencing labs to minimize the effect:
>- Long Ranger gels are reported to suffer less from these
>artifacts.
>- avoid tap water when washing your glass plates.
>- The hotter the wash water, the less buildup you get.
>- various detergents are reported to help. You might
>try 'MultiTerge', available from Baxter Scientific.
>None of these is a complete solution. We have implemented all
>of them, and have reduced the frequency of fades/smears quite
>considerably, but we still occasionally get them.
>I would be MOST appreciative to hear of any real solutions
>to this problem. I am tired of seeing large numbers of samples
>(and large amounts of money) blown away by these artifacts.
>
>Bob Lyons
>University of Michigan
>
>-----------------------------
>Robert Lyons, Ph.D.
>Director, DNA Sequencing Core
>University of Michigan
>-----------------------------