T4 kinase labelling efficiency
logand at msdos.montpellier.inra.fr
Mon May 30 06:21:03 EST 1994
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/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
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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