5' labelling with S-35

skspoidn at reading.ac.uk skspoidn at reading.ac.uk
Wed Mar 24 07:32:39 EST 1993


Sorry its taken so long.
re 
----
Does anyone have a protocol that works for 5' end-labelling of
oligos with T4 polynucleotide kinase and gamma-S35-ATP?
---

Well, I dont know why you would want to (use S35 this way), but the 
following protocol comes from the Promega fmol sequencing system...

10 pmol primer
10 pmol s35-ATP
1x T4 PNK buffer
20 units T4 PNK

final volume 10-20 ul

Incubate at 37 degrees for 4 hours. 

I have never done it, but were it me, I owuld also be
 tempted to 'top up' the PNK after 2 hours. 
Of course, 20 units is quite a lot...

Keep up the radiaoctive work! 
non radioactive labelling is for wimps :)

Mike Poidinger
Dept of Microbiology
University of Reading
UK



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