how to degrade oligos?

Paws03 paws03 at mindspring.com
Fri Jul 11 09:22:13 EST 2003


Hi-

I have a reaction that contains a plasmid and an 51-mer oligo.  After
the reaction, I would like to remove the oligo, leaving me with just the
plasmid.  The problem is, the oligo consists of homologous sequence to the
plasmid, and the first and last 6 bases are phosphorothioated (to prevent
degradation during the reaction).  Additionally, the plasmid (which I'd like
to keep) is nicked, during the reaction, so I have to take care not to
degrade it, as well.  I've tried the following:

1.  Miniprep'd with a plasmid miniprep kit
2.  Phenol/chloroform extraction followed by ethanol precipitation
3.  Phenol/chloroform extraction followed by ethanol precipitation.  Then, 
heated the precipitated DNA to 55 degrees Celsius in a denaturing buffer
for 1 hour, and immediately cleaned up with a PCR clean-up kit.

So far, no luck.  Does anyone out there know of a way that I can rid
myself of the phosphorothioated oligo and not degrade my nicked plasmid?  Maybe
something that you would use following PCR??  Any help would be greatly
appreciated!

Staci

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