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?

  • $\begingroup$ 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 '19 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 '19 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 '19 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 '19 at 0:24
  • 1
    $\begingroup$ A much simpler way to insert sequences that are really small is to order the DNA as oligos and anneal the complementary oligos such that they have compatible overhangs with the overhangs from your restriction digest $\endgroup$ – pepsiandsoda Oct 2 '19 at 20:58

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.


Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.