A short linker

S. Findley polliwog at u.washington.edu
Sun Oct 26 16:09:01 EST 1997


Well I would suggest the inverse strategy to the one suggested in the
previous posting:
Namely, I would leave the annealed oligo pair that you want to insert
*Unphosphorylated* and do the ligation with simpy digested target
construct. In this way you avoid the (very high) probability of cloning
multimers of your oligo. Especially if you can decide to have two
different sticky (cohesive) ends to clone by. (E.g. EcoRI and HindIII)
target vector self-ligation would not be a problem then, and you will only
get a single insert in there. Species that have annealed with multimers of
your oligos in the ligation will *not* be ligated (to form multimers) if
the oligos are unphosphorylated.

And even if you only want to insert using a single type of ovrhang (EcoRI
for instance) if you do your ligation with a molar excess of annealed
oligos you will get what you want surprisingly frequently (I use 20:1)


Good luck.


-SDF
-- 
Seth D. Findley               polliwog at u.washington.edu
Department of Biochemistry    Phone: 206-543-1710
Health Sciences Building J579
University of Washington, Box 357350, Seattle, WA 98195



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