Oligo purification via urea gel??

Ashok Aiyar ashok at biochemistry.cwru.edu
Tue Apr 6 22:49:52 EST 1993


In article <1pt7jh$siv at usenet.INS.CWRU.Edu> txt15 at po.CWRU.Edu (Tao Tao) writes:

>I have encountered problems with a sequencing primer which gave doublet bands.
>It seems to me that this is because of the inpurity of the oligo, a mixed
>population with two species differ by one base.

Very unlikely.  If the length difference were at the 5' end (expected as
oligos are synthesized 3' to 5'), the one base difference should not cause
the doublet during a sequencing reaction.  If there is a problem at the 3' 
end, the "missing" base would simply get filled in during the sequencing 
reaction.

If you had doublets after a straight-forward primer extension, it is likely
that you have two species in your oligo preparation.....

>What I would like to know is whether it can be purified if what I said above is
>true.  I would be grateful if some one in the net land can provide me with
>the protocol for doing this.

I routinely purify all of my oligos on PAGE-UREA gels.

oligos < 30 bases on a 20% gel, 7M urea.  I visualize them by UV shadowing,
cut, and macerate the band, and extract overnight in 0.5M Ammonium Acetate 
with 50 mM EDTA.  The next day, I pass the eluate over a C-18 column, and 
elute the oligo off the column using 50% methanol, and dry it down in a 
SpeedVac.

For oligos > 30 bases I use a 15% gel.

If you want a more detailed procedure, give me a call at x3300.

Ashok

--
Ashok Aiyar                        Mail: ashok at biochemistry.cwru.edu
Department of Biochemistry                       Tel: (216) 368-3300
CWRU School of Medicine, Cleveland, Ohio         Fax: (216) 368-4544



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