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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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  #1  
Old 07-10-2003, 09:06 PM
Paws03
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Default how to degrade oligos?



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
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  #2  
Old 07-10-2003, 09:20 PM
EK
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Default how to degrade oligos?


"Paws03" <[Only registered users see links. ]> wrote in message
news:2938068.1057871180869.JavaMail.nobody@wamui02 .slb.atl.earthlink.net...
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:
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.
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!

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


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  #3  
Old 07-10-2003, 10:46 PM
Tom Knight
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Default how to degrade oligos?

A sephadex size exclusion column should do this job easily. You
might have to experiment a little to figure out the correct version.
G-25 would be a good initial guess.
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