Dear Sequencers and Genescanners,
There have been several requests for assistance with gel problems on
ABI377s recently on the newsgroup. We have recently come through a very
trying time with similar problems and here is a solution that works for
us.
PROBLEM
Something is sticking to the plates that affects the migration of the DNA.
It causes a loss of resolution, stretching and retardation of bands over
150 basepairs. On slightly affected plates the sequence will come good
after 300 basepairs. The distortion is affected by the concentration of
DNA, more DNA less distortion.
RESULT
Sequencing gels that only give 200 bp even with strong signal.
Genescan gels with standards over 150 bp missing or reduced signal for 250
and 300 bp standards. Genescan gels with 2 sets of peaks in the same lane
for standards over 150 bp. They differ by about 2 bp.
SOLUTION
Soak them at least 30 min in 2M NaOH and then 20 minutes in 2N HCl to
neutralise them. Then rinse them thoroughly in water and wash them well
with alconox. THIS ROUTINE PROCEDURE HELPS BUT DOES NOT MAKE PLATE
PERFECT.
We have to also wash twice in ethanol scubbing each time very hard with
lint-free tissues then polish the plates with tissues using lots of elbow
grease. We have to do this procedure everytime we run a gel or the
problem
returns.
If anyone knows what the cause is we would like to know. Prevention is
better than cure. We do not touch plates with gloves because that was a
suspected cause. I thought it might have been the Sydney water but it
seems to be a world-wide problem. I wonder if it is the Alconox?????
Best of luck all,
Alan Wilton
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Alan Wilton
School of Biochemistry and Molecular Genetics
University of New South Wales
Sydney NSW 2052
Australia
Phone +61 (2) 9385 2019
Fax +61 (2) 9385 1483
Email a.wilton at unsw.edu.au
