Steve Stelman (steves at burn.ucsd.edu) wrote:
> I have successfully annealed short oligos (14 bp) and ligated these into plasmid with
> complementary overhangs without purification from ss molecules. The procedure that has worked
> for me is:
> 1) quantify the oligo's and make a mixture of equal molar amounts of each oligo (or equal conc.
> when both oligo's are the same length)
> 2) make sure everything is denatured by heating mixture 10 min in 70C water bath (more than
> 3) transfer 70C water and tube containing mixture to styrofoam box (like those used to ship
> 4) cover box and let cool to RT to anneal
> I've then ligated with T4 DNA ligase, using molar excess of oligo linker (not purified or
> quantified). I have observed concatemer insertions of oligos at times, but design of oligos with
> unique sites internally or at each end enables removal by restriction digest.
> Hope this helps
If the oligo's are not phosphorylated, they will not form
concatamers, instead you will get one ds linker on each end of the
plasmid. One strand of each will be ligated, and the other gapped.
Re-heat the linker-tailed plasmid, and slow cool as above, and the circle
will close with exactly one linker inserted into the plasmid. The
non-ligated, gapped strands will be repaired after transformation. This
has worked several times for me.
Michael J. Corboy
Molecular and Cell Biology
University of Texas at Dallas