T4 kinase labelling efficiency

LOGAND logand at msdos.montpellier.inra.fr
Mon May 30 06:21:03 EST 1994

Dear All,

I am labelling an oligo with 32P using Gibco T4 kinase. Although I use a 4 fold 
excess of ATP over the oligo concentration I only get a maximum of 30% of the 
oligo labelled. 
Mixture contains 5pmoles oligo, 25pmoles ATP, 20 U/ul T4 polynucleotide kinase.
I perhaps assume incorrectly that the binding to DE-81 filters is not the cause 
(ie losing labelled oligo because it is not bound tight when I wash the 
filters). I read that with an excess of ATP a smaller percentage of the label 
would be transferred but much of the oligo would be labelled which is what I 
want, however it hasn't worked. Am I using too low a concentration of ATP? 
Perhaps I am being unrealistic - are those figures of near 100% of oligo 
labelled unobtainable?

I would appreciate comments from other users of this method and any suggestions 
to improve yield.

David C. Logan
Logand at montpellier.inra.fr  

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