Subcloning with oligos

douglas l feinstein dlfeins at cumc.cornell.edu
Fri Jan 21 17:52:38 EST 1994


Subject: Subcloning with oligos
From: bates, dbates at hph.ucdavis.edu
Date: 21 Jan 1994 21:03:37 -0000
In article <2hpfv9$mdh at mserv1.dl.ac.uk> bates, dbates at hph.ucdavis.edu
writes:
>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


PCR it!!!

doug



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