Methylation protection- doublets using 3' ddNTP labeling

Paul Mitsis pmitsis at seqltd.com
Tue Jan 18 11:47:49 EST 2000


Peoples,

       I am running methylation protection/interference assays using a probe
labeled on the 3' end with a32P-ddATP. It seems that after the reactions
(protection, piperidine cleavage, workup etc.) that the low mw bands
corresponding to <15 bp run as doublets. This is also very prominent in the
A+G Maxim/Gilbert formamide reaction run for a standard.  I haven't seen
this before with 5' end labeling with PNK.  Does anyone know if  there is a
small charge heterogeneity in the reaction products of this method
introduced by using a 3' ddATP label?  Any help would be greatly
appreciated.

Yours,

Paul Mitsis Ph.D.
Senior scientist
Praelux Inc.






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