Sephadex G-50 problem

Richard Friedman rfriedma at fhcrc.org
Thu Dec 17 22:02:16 EST 1998


> At 17:56 12/16/98 -0700, Long-Cheng li wrote:
> >Hi, there
> >
> >This is the first I used Sephadex G-50 coloum (Promega) to purify r-32P-ATP
> >labelled oligo probe. After I loaded the sample (10-20ul) and centrifuged
> >for 5min at 1000g, I got the elute that had hardly any counts as detected by
> >a Geriger counter. Even I extended the centrifugation time the result was
> >the same. I don't think my oligo was not labelled at all. Should someone
> >have the experience of this kind, Please give me a reply. Thanks
> 
> Long:
> 
> If memory serves (and the BMB Catalog), G50 has an exclusion limit of about
> 70-72 bases.  So unless you're oligo is 75-80 bases, chances are it is
> still in the column along with your unbound 32P.
> 
> For oligos, I always use G25 that has an exclusion limite of about 12 bases
> (single stranded).  It is best suited for oligo labeling.  
> 
> As with a previous post, precipitation is an option but I've used the G50
> and G25 for both DNA and RNA work without problems.

Actually, If memory serves me, I used to use g50 spin columns to clean up
after endlabeling 20mers.



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