Emma Berta Gutierrez
egutie at IFCSUN1.IFISIOL.UNAM.MX
Thu Aug 3 12:50:50 EST 1995
Here are all (or almost all) the recomendations I received from the
Chlamy netters to avoid DNA compressions. Thanks a lot to every body out
I.- Having single-standed templates and using deaza-dGTP in the
sequencing reactions gets through almost all compressions. You can use
dITP but it isn't as good as deaza.
From: David R. Mitchell.
II.- Using TdT helps more to eliminate false stops than compression, but
it REALLY cleans up the reactions, you can use it as in the USB comments
article: Artifact Banding when sequencing double-stranded DNA templates
by T.W. Fawcett and S.G. Bartlett.
Also, constant temperature gels are recomended to eliminate compressions
in GC rich DNA.Temperatures close to 55 degrees (celsius).
From: Julie Knott.
III.-Using sequenase and standard TBE/urea buffer gradient gels also was
another advice. Reading problems will also be present (with ss DNA) due
to enzyme stops (dark bands in all four lanes), in this case Taq
Polimerase won't help.
From: Dave Mitchel.
IV.- You can use formamide in gels (30% v/v), the advice was to buy it
from Fisher Biotech, the "molecular biology grade" (kept it at -20 until
used). Maniatis describes how to deionize it.
The gel composition was: 8M urea, 1x TBE, 30% formamide, 6% "acrylamide"
(from a "30% acrylamide stock": 28.4% acrylamide + 1.6 % bis).
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