General 373 and 377 problems

Shaun Tyler styler at hpb.hwc.ca
Thu Aug 1 07:05:46 EST 1996

> Date: Thu, 11 Jul 1996 10:58:05 -0500
> From: keller at email.chop.edu
> Subject: Re: General 373 and 377 problems
> I would like to get any suggestions or input from those of you running
> 48
> cm plates on either your 373 or 377. We are having two separate
> problems
> that have resulted in much lost data and wasted time.
> First, on our 373, since switching to the longer 48cm plates, we have
> had
> intermittent problems with stoppages in electrophoresis. Some of these
> seem to be due to leakage behind the plates, resulting in a lower top
> buffer level and an increased bottom buffer level. Other times, the
> amounts of buffer in both chambers looks unchanged, but there is
> evidence
> of buffer leak down the spacers (salt ppt on the ear area of the
> spacers).
> We have tried replacing the gasket, installed force-limiting screws on
> the
> upper buffer chamber, under-tightened and over-tightened the buffer
> chamber, taped the ears during polymerization, etc. At this point we
> are
> considering the possibility that the problem may be due to the design
> of
> the system leaving the top of the ears unclamped and accessible for a
> small amount of buffer to cause a short circuit when it runs down the
> spacers. Has anyone else had a similar problem? The result is that we
> get
> short runs of 120-350 bases depending on when electrophoresis stops.
> Even
> if we happen to be around when it happens, it seems to be irreverible.
> Second, on our 377, with long plates, we get noisy plate checks even
> when
> we have stripped our plates (thanks for all the suggestions on
> stripping
> methods), avoided using any kind of cleaner on the plates, switched to
> Amresco reagents etc. Even if we load lanes that look clear on the
> plate
> check, they still end up showing background fluorescence (green or
> yellow)
> that mutes the samples and makes the data useless. It has been
> suggested
> to us that the placement of the cassette in the machine is tricky with
> the
> larger plates and may result in plates that are not aligned correctly
> in
> relation to the lasar. Anyone with similar problems or suggestions on
> how
> to correct this problem?


I may have a part answer to your first question.  When we first started using the 373's we also had  problems 
with buffer leaks.  It turned out that the gel was not always polymerizing where it contacted the spacer.  As a 
result the buffer slowly leaked down the channel formed.  To get around this problem we started pushing the top 
of the spacer into the gel before tightening the outer bracket.  This distorts the top of the gel somewhat but 
since only the centre region is used it doesn't affect the running area.  I hope this helps and good luck with 
your other questions.

Shaun Tyler
DNA Core Facility
Laboratory Centre for Disease Control
Health Canada

Ph#:  (613) 941-6441
FAX#: (613) 957-1358

E-mail:  styler at hpb.hwc.ca

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