We had the same problem for quite some time. To finally fix it, we
switched to the Sakabe water run in method. You can get the protocol at
www.pebio.com. We actually run the DNA in with a .5xTBE solution in the
top buffer chamber and add 10x to bring it up to a 1xsolution. For the
longest time we thought that we were disturbing the gel (and the odds) when
we loaded the evens, but no matter how careful we were, we still got the
problem. The only thing that I can correlate to the onset is the addition
of a new water purification system that we installed at about the same time
as the appearance of our problem.
The downfall to the sakabe method is that the salt front from the
loading buffer will often migrate at the same rate as the first 5-10bp so
you might loose these in some runs. This is also sporadic and we are
trying to design a protocol that will abolish this problem.
I hope this works, I know how frustrating it was when our lab was
experiencing this.
-Brian
THE DNA SEQUENCING SERVICE
UNIVERSITY OF ARIZONA
(520)621-9184
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Brian Coullahan- coullaha at u.arizona.edu
Heather Glanzberg- heatherg at u.arizona.edu
Janet Barnard- jbarnard at u.arizona.edu
Svetlana Reznikova - svetlanr at u.arizona.edu
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