"Joseph C. Bagshaw" wrote:
> Your working way too hard. As others have suggested, just design the
> desired sticky ends into your oligos. If you don't dephosphorylate your
> cut vector you won't even need to phosphorylate your oligos. With 40-mers
> you couldn't keep 'em apart with a crowbar, even at room temp. Just mix
> your oligos in ligase buffer, add your cut vector and a pinch of ligase,
> and follow your favorite ligation and transformation protocols. Been
> there, done that.
I agree with that. Moreover, if you don't phosphorilate your oligos you
are sure of not getting multimers in the cloning step. So you can add
high concentration of your annealed oligo to increase the ligation
efficiency without worrying about tandem cloning.
Divison of Genetics
University of Alicante (Spain)