35S-labelled oligo purification

Danny McLaughlin D.P.McLaughlin at mds.qmw.ac.uk
Wed Feb 2 11:10:58 EST 2000


> There is a lot on Terminal transferase and labelling oligos with various
> dNTPs and buffers using Mn and Co in Methods in Enzymology, vol. 100 pp
> 96-116.

I will try to get a hold of this.  Thanks.

> If the interaction is 'ionic' then can you not equilibrate the column in
> a buffer with a little salt, doing the same with the oligo sample. The
> only difference should be the unincorporated dATP which with luck will
> be retained as part of the desalting process whilst the labelled oligo

Tried salt equilibration up to 150 and then 500 mM, then STE and STET and
NOTHING seemed to do the trick.  Basically we have decided that these
columns are not useful for this purpose (35S labelled oligos) and will steer
clear of them - I advise other to think carefully about using them too!

> How much salt? Not sure but maybe 25-50mM NaCl or KCl would be enough as
> a G25 should have minimal ionic binding. Whether you then have to get
> rid of salt for a subsequent procedure could then be a problem but hey
> that's research!

Now we have the problem of finding that extra 200-300 pounds for more
35SdATP and enzyme.  But hey, unfortunately, that's research! :-)

Cheers,

Danny

>
> Duncan
> --
> The problem with being on the cutting edge is that you occasionally get
> sliced from time to time....
>
> Duncan Clark
> DNAmp Ltd.
> Tel: +44(0)1252376288
> FAX: +44(0)8701640382
> http://www.dnamp.com
> http://www.genesys.demon.co.uk






More information about the Methods mailing list