35S-labelled oligo purification

Danny McLaughlin D.P.McLaughlin at mds.qmw.ac.uk
Thu Feb 3 07:11:56 EST 2000


Dr. Duncan Clark <Duncan at nospam.demon.co.uk> wrote in message
news:Zpn4$JAYNGm4EAYk at genesys.demon.co.uk...
> In article <879l1j$ahl$1 at beta.qmw.ac.uk>, Danny McLaughlin
> <D.P.McLaughlin at mds.qmw.ac.uk> writes
> >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!
>
> Could it be that as you have tail lengths of maybe 50-100bp that these
> are getting into the pores of the sephadex beads and wrapping themselves
> around the bead. The dATP is supposed to get slowed down by migration
> through the pores whilst the larger molecule goes around so what I am
> suggesting looks wrong. However if the tailed oligo gets trapped this
> way the vortexing may free them as you are finding (maybe by shearing?).

We start with 4 pmols of variable length oligos (say 40-mer) and label with
50 microCi of 35SdATP (1250 Ci/mmol).  This means that we have a 10-fold
molar ratio of label to oligo.  So it's impossible to label up to 50-100
tails.  With 30% incorporation (our usual), we reckon most oligo molecules
have maximum 3-4 tails each.  So i doubt if a 45-mer oligo (including tail)
would be sheared by vortexing.  I'm not certain, though.  Pharmacia
acknowledge that there is a problem with these columns for 35S - they just
fail to tell anyone about it until they ask!

> There again who cares about the research if you need to get the money
> for new label and you if you have better method anyway!

Eh? :-)

Danny

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Dr. Danny McLaughlin
Anaesthetics Unit
St. Bartholomew's and The Royal London School of Medicine & Dentistry
The Royal London Hospital
London E1 1BB
Email:  d.p.mclaughlin at mds.qmw.ac.uk
URL:  http://www.mds.qmw.ac.uk/anae/neurolab.htm
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