Oligonucleotide Labeling with Polynucleotide Kinase

Caroline Szymeczek-Seay css at med.unc.edu
Tue Sep 28 13:42:09 EST 1999


mlysik at hsc.vcu.edu wrote:
> 
>         I am trying to label my oligonucleotides using gamma-32P-ATP and
> polynucleotide kinase.  However, the labeling efficiency I get is low (often <1%) and I
> get a high amount of incorporated label following purification. 

This is probably obvious, but are your oligos cleaned up?  Ammonium ions
(present in the solution used to cleave oligos from synthesis columns)
will inhibit the kinase reaction.   I don't find that a lot of
purification is needed, but I do ethanol precipitate and do 2 70%
washes.  This is a moot issue, of course, if your oligos are already
somewhat purified.

caroline
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