wgschech at med.uni-tuebingen.de
Tue Sep 1 04:11:48 EST 1998
Seems to be a question a of temeprature. At least I observed this
when pre-running sequencing gels too long. The cause probably is
expansion of the gel.
You might try running the gel at lower temperatures (=less voltage)
or in the cold room. If it's possible to decrease the mechanical
force that clamps to two glass plates together in the loading region
of the gel, this could help, too.
Just some ideas.
> We are trying to use taurine gels for manual sequencing with new
> thermosequenase and ATP-p33. We got some trouble in the loading
> surface after 2 hours run at 50oC, that seemed to be uneven and
> irregular, making the second loading impossible. Conditions used
> for the gel: 6% acrylamide, 8M urea, 1 X GTG buffer, 1 ml APS 10%,
> 25 microlitres of TEMED (for a 100 ml gel). All itens are from
> Pharmacia except for GTG buffer:USB mixed powder . These conditions
> were suggested at the thermosequenase protocol by Amersham. We also
> tried TEMED and APS 25%, 100 microlitres each, and the problem
> Does anybody would have a suggestion to overcome those problems?
> Please help me! I would appreciate very much any sympathetic
> Mario Mayer
> Instituto de Ciencias Biomedicas
> Universidade de Sao Paulo
> e-mail : tefcosta at usp.br
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