resolution loss at 300 bases

julie feinstein jfstein at amnh.org
Wed Oct 21 04:54:52 EST 1998


Hi,

I'm having a puzzling problem with dRhod terminator sequencing gels on an
ABI 377. My sequences start clean and bright, get to about 300 bases and
quickly become stretched out and unusable. I notice that the top of the
gel, tho flat when loaded, becomes distorted and uneven after only a few
minutes into the run. It swells into the wells at places and pulls away
from them in others. At times the gel starts to get ragged and deformed
during the prerun. I have tried loading the gel at 35 degrees after a 15
minute prerun to prevent the wells from heating and degrading but this is
not a dependable cure.

Other users here are sequencing up to 800 bases, and never see this kind of
problem. We all use the same reagents, plates, combs and general protocol.
Others have sequenced DNA that I prepared without problems -- I think the
template is ok.

I suspect some subtle problem with gel construction. If anyone reading this
has had this problem and can suggest a possible solution or avenue for
exploration I would appreciate suggestions.

Thanks very much.

J. Feinstein





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