General 373 and 377 problems

keller at email.chop.edu keller at email.chop.edu
Thu Jul 11 10:11:30 EST 1996


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?

Thanks in advance for your responses!
Please email me at keller at email.chop.edu as well as well as posting to the
group.
Margaret Keller
The Children's Hosp. of Philadelphia
(215) 590-3897




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