End-labeling in gel slices?

John Watson ajwatson at romeo.caltech.edu
Tue May 17 23:29:57 EST 1994


In article <msr2-170594182659 at cu-dialup-0302.cit.cornell.edu>,
msr2 at cornell.edu (Mark S. Rose) wrote:
> 
> Hi folks
> 
> I would like to label the ends (and only the ends) of a 500kb DNA fragment.
>  I'm concerned that extracting this fragment from a gel prior to labeling
> it may result in shearing which presumably would result in positions other
> than the ends also being labeled.  To avoid this I'm thinking about
> performing the end-labeling in low melting point agarose.  Does anyone know
> how effective T4 polynucleotide kinase is in agarose or have other
> suggestions as to how I might achieve my purpose.   
> 		                                       Thanks
>                                           Mark

It's been my (perhaps limited) experience that most every enzyme is active
in LMP agarose -- and I've done random priming, ligating, restriction
digests, but not, as I recall, end-labeling.  Still, I'd have no qualms
about it.  FMC puts out a couple of nifty little booklets describing all
the things you can do in Seaplaque and Nusieve GTG agaroses -- yours for
the asking from FMC (and no, I am not affilliated etc etc).

John



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