Subcloning oligos into expression vectors

David H. Gorski d-gorski at uchicago.edu
Mon Jan 20 12:07:45 EST 1997


> I have done the same thing without any problem. Anneal the oligos by
> heating the mixture for 2 min in a boiling waterbath and slowly cool
> to RT. Cut your vector, add your annealed oligos in a 100:1 ratio to
> your vector, ligate i hour RT and cut your ligation mixture with Nhe I
> and BglII ( I assume that the insertion of your oligo destroyes the
> restriction sites) and transform. Your background is probably the
> result of inefficient cutting of one or both enzymes resulting in the
> selfligation of the single cut vector. 

Thanks for the tips. I would modify the suggestion and cut only with Nhe I,
because the Bgl II site is not destroyed by ligation, although the Nhe I
site is. The reason for this is so that I can cut with Bgl II again and Spe
I (a site on the insert) and drop another copy of my insert into that site
(Nhe I ends are compatible with Spe I ends), thus producing a doublet. I
can then do it again to produce a triplet, and so on.

Of course, I am getting ahead of myself. I have to produce a singlet
insert, first, before going wild with making multimers.... :(

-- 
David H. Gorski, M.D., Ph.D. |"What is man, when you come to think
Department of Surgery        | upon him, but a minutely set, ingenious
University of Chicago        | machine for turning, with infinite
                             | artfulness, the red wine of Shiraz
E-Mail: d-gorski at uchicago.edu| into urine?"--Isaak Dinesen



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