Polynucleotide Kinase At pH 9.5

pgegen at rnaworld.bio.ukans.edu pgegen at rnaworld.bio.ukans.edu
Fri Oct 6 17:56:51 EST 1995

In <451c32$gi5 at tribune.usask.ca>, goldie at duke.usask.ca (Hugh Goldie) writes:
>Several years ago, an article was posted recommending pH 9.5 for 
>polynucleotide kinase reactions and referring to an article in the 
>literature (I think it was in an old issue of Analytical Biochemistry).
>I was unable to find the journal article via Medline.  Also I could not find
>the old message in the archives of bionet.molbio.methds-reagnts.  (I found
>Can anyone send me the reference or a copy of the news group message?
>Hughes Goldie, University of Saskatchewan, Canada
>goldie at duke.usask.ca

I don't have that reference, BUT I can tell you (1) that I and others have done PHK reactions in Tris buffer at pH 9.
(2) Harrison & Zimmerman, NAR 14, 1863-1869 (1986) and Harrison & Zimmerman, Anal. Biochem. 158, 307-315 (1986) 
described optimum conditions for the EXCHANGE labelling reaction  of PNK. These are
	50 mM Imidazole-HCl, pH 6.4
	4.5 mM DTT
	18 mM MgCL2
	0.1 mM ADP
	6% (w/v) PEG-8000
	PNK as needed
Incubate 30min at 37C.  (The PEG stabilizes the PNK ca. 10-fold.)

| Peter Gegenheimer                          |  pgegen at kuhub.cc.ukans.edu    |
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