35S-labelled oligo purification
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Wed Feb 2 10:24:53 EST 2000
In article <879dtv$7b8$1 at beta.qmw.ac.uk>, Danny McLaughlin
<D.P.McLaughlin at mds.qmw.ac.uk> writes
>> Am I missing something here? How are you labelling oligos with 35S dATP?
>> Are you not kinasing using T4PNK and gamma labelled 35S ATP?
>Nope, 3' end labelling with terminal transferase. 5' tailing doesn't give
>a hot enough probe for in situ hybridzation.
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
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
goes straight through. The addition of salt should not interfere with
the removal as it will be common to all buffers but may overcome the
interaction. Of course if the interaction is instead hydrophobic then
salt is the last thing to try. In that case 20% ethylene glycol may be
better, or dare I suggest it a few M urea!
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
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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