correction: T4 kinase labelling efficiency
logand at msdos.montpellier.inra.fr
Mon May 30 09:17:19 EST 1994
change - 20 U/ul to 20 U.
Sorry for error.
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
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
I would appreciate comments from other users of this method and any suggestions
to improve yield.
David C. Logan
Logand at montpellier.inra.fr
More information about the Methods