Subcloning with oligos

bates dbates at hph.ucdavis.edu
Fri Jan 21 16:03:37 EST 1994


I've been trying to put a small (25bp) polylinker into an expression vector,
and having some problems. I've made the polylinker from two complementary 25bp
oligos, with HindIII sites at either end. The oligos, when annealed should make
up a polylinker with three restriction sites, and a false HindIII site. I've
kinased the oligos, cut the vector with HindIII, phosphatased the cut vector,
gel filtration treated the kinased oligos, annealed the oligos, and tried to
ligate them in. All the controls work (religated cut vector :-), religated
phosphatased vector :-(, uncut vector :-) etc) but I don't get any colonies
when I try and ligate in the insert.

Has anybody experienced these problems when trying to use oligos in subcloning?

Thanks

Dave



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