Tammi-
I am not familiar with the Genomic Solutions set-up, but one (perhaps
remote) possiblity is that the upper chamber is filled too high, thus
allowing for the buffer to be drawn up at the edges via capillary attraction
leading to a slow but eventual depletion of the upper buffer. I've seen
this happen with the BioRad Protean II system, and the fact that your "gels
hold buffer until they are placed into the gel box with/ 3L of buffer"
supports this idea.
David
__________________________________________________
David B. Friedman, Ph.D. (303) 315-4712
Dept. of Cellular and Structural Biology, "I am not a number,
University of Colorado School of Medicine I am a Friedman!"
UCHSC, B-111, SOM 4538
4200 E. Ninth Ave.
Denver, Colorado 80262
-----Original Message-----
From: Kathryn Weiss [mailto:kcweiss@ucdavis.edu]
Sent: Thursday, February 15, 2001 5:03 PM
To: Recipients of ABRF List
Subject: Leaky 2D gels
I am posting this for a colleague in my lab. Replies can be sent directly
to her at <tlolineka@ucdavis.edu>, if desired.
Our lab has recently started pouring our own second dimension gels
(approx. 9"X11") using the Genomic Solutions system. The gels polymerize
well and hold buffer overnight; however, buffer from the upper chamber leaks
into the lower chamber during the second dimension run. The buffer is
actually
leaking between the edge of the gel and the spacers, and possibly between
the glass and the gel surface. It is a slow leak, but enough to create
problems with the run.
There is no visible channel between the gel and the spacer. The spacer is
flush with the glass plates, & the gels are poured to about 1cm from the top
of the gel. There is no visible leak on the outside of the plates or by the
gasket.
I've tried a variety of things and nothing has stopped the leaking. I've
tried using different amounts of glue (stick), different spacers, making an
agarose plug, hot glueing the sides of the plate, and several other
things. I have even had other people pour gels to see if my technique is
the problem. They have had the same problems.
The gels hold buffer until they are placed into the gel box with/ 3L of
buffer
and a current running. This has affected protein migration, running time,
and (obviously) reproducibility.
If anyone has had similar problems, I welcome your suggestions. Thank you.
Tammi Olineka
<tlolineka@ucdavis.edu>
Department of Viticulture and Enology
University of California, Davis
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