how to degrade oligos?

EK nobody at elnino.com
Thu Jul 10 16:20:53 EST 2003


"Paws03" <paws03 at mindspring.com> wrote in message
news:2938068.1057871180869.JavaMail.nobody at wamui02.slb.atl.earthlink.net...
> Hi-
>
> I have a reaction that contains a plasmid and 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
> ---

Can you run the mixture on agarose gel and then purify the desired band
using conventional gel purification kits?
- Emir





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