Eliminating DNACompressions

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 
there!.
Ember

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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