Sephadex G-50 problem
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
> 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.
More information about the Methods