correction: T4 kinase labelling efficiency

LOGAND logand at msdos.montpellier.inra.fr
Mon May 30 09:17:19 EST 1994


change - 20 U/ul to 20 U.
Sorry for error.

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 T4 polynucleotide kinase, 
20uL volume. 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 
Montpellier
Logand at montpellier.inra.fr




More information about the Methods mailing list