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how to degrade oligos?

how to degrade oligos? - Protocols and Methods Forum

how to degrade oligos? - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 07-11-2003, 02:22 PM
Paws03
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Default how to degrade oligos?



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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