rhubner at molbiol.ox.ac.uk wrote:
>> Dear bionetters,
>> anybody out there ever annealed short oligos (10-13 bp with one or two 4-base
> overhangs; hence, 6 or 9bp overlap "only") and purified them from ss
> I think ligating with the mix (unpurified) will scramble up the rx'n...
> alternatively, I might try ligating the ss-molecules with T4 RNA ligase and
> somehow anneal and hope for the bacteria to repair...
>> Any advice/empirical info would be HIGHLY appreciated!
> Thank you sooo much & Merry Xmas to you all!
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