5' end-labelling with S-35-ATP
andersen at cs.uwp.edu
Thu Mar 25 13:55:27 EST 1993
tlyoung at kean.ucs.mun.ca wrote:
: Does anyone have a protocol that works for 5' end-labelling of
: oligos with T4 polynucleotide kinase and gamma-S35-ATP?
: Terry-Lynn Young Fax: (709) 737-7010
: Memorial University of Newfoundland
I follow a protocol that was supplied by USB with their TAQuenase DNA
sequencing kit. They recommend the following for labelling 20 pmol of
primer in a 10 uL volume:
- using 20 units of PNK (rather than 10 units as for P32).
- not using more than 30 units (this results in no labelled product).
- using 25 pmol of gamma-labelled S35 ATP (2.5 uL of 1200 Ci/mmol;
- incubating at 37C for 2 hours (rather than for 10 min as for P32).
- finally inactivating at 95C for 2-5 min.
The 1X labelling buffer is: 50 mM Tris-HCL pH 7.6
10 mM MgCl2
10 mM 2-mercaptoethanol
I have used this protocol once for labelling a sequencing primer (M13 -40).
In my miniprep sequencing reactions (using about 2 ug ds plasmid DNA) I used
twice the amount of primer recommended and cycled my reactions 25 times.
This resulted in a readable sequence after a 3 day exposure of my
fixed/dried sequencing gel. Overall it worked well, although the bands
were slightly less intense than I expected them to be. Next time I think
I'll just cycle longer.
By the way, the TAQuenase kit was a FREE SAMPLE from USB ( $185.00 retail )
and the enzyme and reaction buffer was supplied with that kit.
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