Subcloning with oligos

Helge Weissig helgew at ljcrf.edu
Fri Jan 21 17:31:47 EST 1994


Dave Bates <dbates at hph.ucdavis.edu> wrote:
>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


yes, unfortunately :), but the solution was rather easy (and obvious), it
was simply the ratio of annealed oligo to vector. I was using way too much
oligo at first, and this seemed to inhibit the ligation or something like
that.

Did you check your oligo for satisfactory annealing (gel electrophoresis)
before the ligation?

and... do the oligos have the right sequence (no kidding! i lost a whole
two weeks on a simple bug like that, and only found out, b/c a co-worker
told me that i had put down the wrong sequence in the order sheet)

good luck!
helge


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Helge Weissig      

La Jolla Cancer Research Foundation             (619) 455 6480 x253
10901 N. Torrey Pines Rd.                  FAX: (619) 455 0181
La Jolla, CA 92034                      e-mail: helgew at ljcrf.edu
USA

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