35S-labelled oligo purification

News D.P.McLaughlin at mds.qmw.ac.uk
Wed Feb 2 05:37:19 EST 2000


Dear Hiranya

> >We are having trouble purifying 35S labelled oligos using Pharmacia's G25
> >microspin columns.  Basically the labelled oligo just stays on the
column.
>
> How do you know you have produced oligos? If your label is left behind in
> the column, chances are that the counts are from the dNTP rather than from
> the oligo. I have not used those "microspin" columns, but I do use my
"home
> made" G50 or G25 spin columns (1mL syringe).

The oligos are HPSF pure synthesised by MWG biotech - no problems in the
past and EtOH
precipitation works well - just looking for a quicker method.  Pharmacia
(Amersham)
assure us that the unincorporated 35SdATP will wash off the column, but that
the
interacton with the 35S labelled oligo is 'ionic'.  Trouble is, we can wash
off the
unincorporated 35S after a few washes, but the labelled oligo sticks until
vortexed
vigorously and then eluted.  It is all very puzzling since the product
guides for all
their microspin columns suggests G25 for labelled oligos

> Another possibility: The incorporation of 35S[dATP] (I assume that's what
it
> is) is such that you will not be able to detect it in the eluate with a
hand
> held (ionization) counter, while the column may still be "hot". As a rule,
> the % incoporation is rarely above 25. Are you checking your eluate by
> scintillation counting?

Yes we are using scintillation counting so we know that if all that is on
the column
is free 35SdATP, we only have 3% incorporation - much lower than we usually
achieve
(usually 25-35%)

AmershamPharmacia are dodging the issue - beware of using these columns.

Danny

****************************************************************************
*********
Dr. Danny McLaughlin
Lecturer in Neuropharmacology
Anaesthetics Unit
Division of Surgery, Clinical Neuroscience & Intensive Care
St. Bartholomew's and The Royal London School of Medicine & Dentistry
The Royal London Hospital
Whitechapel
London E1 1BB

Tel:   +44 (0)207 377-7725 or 377-7000 ext 2292 (answerphone)
Fax:  +44 (0)207 377-7126

Email:  d.p.mclaughlin at mds.qmw.ac.uk
If mail is bouncing, try dmclaugh at hgmp.mrc.ac.uk
URL:  http://www.mds.qmw.ac.uk/anae/neurolab.htm
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