how to degrade oligos?
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...
> 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
Can you run the mixture on agarose gel and then purify the desired band
using conventional gel purification kits?
More information about the Methods