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?
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 Legend: __ 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.