What is the cause of "lane skewing" during sequencing electrophor

Miller, Mark J. (MILLERM@dc37a.nci.nih.gov)
Fri, 30 Jan 1998 09:27:03 -0500


We run a small sequencing core facility that accepts samples from various
users. Usually
the user has run the sequencing reaction and we run the samples on an
ABI377, then
post process the data and return it to the user.

Lately we've been getting a lot of samples that case the lanes of the gel to
be skewed
toward the center of the set of samples on a gel. This seems to be a sample
problem
since, with mixed sets of samples on the same gel, the major skew will be
toward the
center of one group. Frequently the problem is very severe, requiring much
manual editing
and loss of data at the beginning of the run.

I've suggested the cause is excess salt in the samples, resulting in
increased conductivity
within the wells of that set of samples, and so skewing the electric field
and hence the lane
pattern.

Is that sensible? Does anyone have a better idea? What can be done to
eliminate the
problem?

Thanks in advance.

Mark J. Miller