35S-labelled oligo purification

Hiranya S. Roychowdhury hroychow at nmsu.edu
Tue Feb 1 17:58:50 EST 2000


At 03:43 PM 2/1/00 -0000, News wrote:
>Hi
>
>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).

You should check your polymerase activity to make sure that you are
incorporating dNTP's into your oligos. To do that, you may scale down your
rxn and run on a mini acrylamide gel... then expose it for autoradiography.

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?

 


>We have tried everything Pharmacia suggest salt washes (now trying STE and
>STET) and wonder if anyone has any suggestions??
>
>Please email a response if possible
>
>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
>****************************************************************************
>*********
>
>
>


Dr. Hiranya Sankar Roychowdhury
GENE LAB/ EPPWS
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu

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