annealing oligos

jen_the_glick at my-deja.com jen_the_glick at my-deja.com
Sun Feb 13 12:11:11 EST 2000


Thanks to everyone for your help.  I am going to give this strategy a
try!

Jennifer

In article <389562B7.B6A7F3B6 at ua.es>,
  Rafael Maldonado <rmaldonado at ua.es> wrote:
>
>
> "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.
>
> -Rafa
>
> --
> Rafael Maldonado
> Divison of Genetics
> University of Alicante (Spain)
>


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