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I am planning to insert a 45 bp sequence in a vector. After restricting my insert to create compatible sticky ends, I am finding no way to clean it up. Is there any way for cleaning this fragment after restriction or should I proceed without clean-up?

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  • $\begingroup$ Proceeding without cleanup will reduce your efficiency but may still work. Have you tried purifying via gel extraction? A high density gel should allow you to separate the band to some degree. $\endgroup$ – Joe Healey Jan 2 at 15:55
  • $\begingroup$ Hi Joe, I can locate the band in high density gel, but would it be possible to extract 45bp sequence through gel extraction protocol? $\endgroup$ – RKK Jan 3 at 5:13
  • $\begingroup$ I think so, though you may need to ensure you use a column designed specifically for small fragment retention. There are columns for purification of miRNAs, so I’d have thought there might be a column you could source which will do a similar thing for dsDNA, but I am guessing somewhat. I believe there are some old-school chemical techniques for precipitating away melted gel without needing a column too that you could try. You could then concentrate with a rotorvap or heatblock. $\endgroup$ – Joe Healey Jan 3 at 8:13
  • $\begingroup$ “GeneClean” which involves adding powdered glass and potassium iodide solution to an agarose gel slice would work for this. I believe QIAgen sells a matrix like this. The glass is extremely fine, and has to be prepared by boiling in nitric acid. Once upon a time there was a company named Bio 101 that sold a product named GeneClean, but they may have been bought by somebody else. For unknown reasons, nucleic acids will bind to glass in the presence of a chaotrope. $\endgroup$ – mdperry Jan 5 at 0:24
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Thanks all for your comments guys. I tried purifying using PCR purification kit. I could recover 120 ng of purified fragment from 500 ng. It is ok for my further work.

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