5' end-labelling with S-35-ATP

Brian Andersen andersen at cs.uwp.edu
Thu Mar 25 13:55:27 EST 1993


tlyoung at kean.ucs.mun.ca wrote:
: Hello
: 	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;
      10mCi/mL).
   -  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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