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.)

o----------------------------------------------------------------------------o
| Peter Gegenheimer                          |  pgegen at kuhub.cc.ukans.edu    |
| Departments of Biochemistry and of Botany  |  voice: 913-864-3939          |
| University of Kansas                       | FAX  : 913-864-5321           |
| 2045 Haworth Hall                          | "You can't fool reality."     |  
| Lawrence  KS  66045-2106                   |                   R. Feinman  |  
o____________________________________________|_______________________________o




More information about the Methods mailing list