I have ordered 36bp oligonucleotides that anneal to each other and create sticky ends to be cloned in a vector afterwards. I have tried cloning many times with different methods and I failed. Now I started to think that my oligos are actually shorter than 36 nt since desalting is not an effective method and most of my oligos do not create complete sticky ends when they anneal to each other. I believe I have also found support for this. So, how much is it possible that most of my oligos are actually shorter than 36 nt ? Is this a good troubleshooting or 36 nt oligos not being purified with more efficient method should not really create a problem?

  • $\begingroup$ I've never had much luck trying to clone something that small into a vector. One thing I prefer to do is to make the oligos long enough to have the restriction sites on each end, with 3 extra bases past those so the restriction enzyme binds better. I cut the oligos, then gel purify, then ligate. But still, it's a small insert. Something that small feels like you could almost PCR it in somehow. $\endgroup$ – user137 Mar 21 '15 at 22:55
  • $\begingroup$ Well, people do ligate annealed oligos directly to cut vectors. Actually it has been said that cloning small inserts is much easier than large inserts but I have no experience on this of course. If you are not familiar with oligonucleotide annealing for ligation and cloning, this may help you: addgene.org/plasmid-protocols/annealed-oligo-cloning $\endgroup$ – ecagl Mar 21 '15 at 23:05

Oligos from well-known companies are pretty good to my knowledge. Problems begin after 100-300bp oligos/dsDNA fragments.

What I suggest is PCRing your vector like that:

          Primer F: ---------------------__________
vector: ======|v site for 36 bp insert v|==========...
        Upstream                        Downstream
        ______----------------- : primer R

__ portion of primer annealing to vector
-- portion that is your insert of interest

Then you can actually just transform that PCR into e. coli, which will seal two nicks together and propagate your vector. Treating your PCR reaction with Dpn enzyme will ensure that all template DNA is removed.


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