Subcloning oligos into expression vectors

Tom Vink tvink at xs4all.nl
Mon Jan 20 07:34:59 EST 1997


d-gorski at uchicago.edu (David H. Gorski) wrote:

>I've been trying to make several of constructs with various promoter
>elements placed in front of a luciferase reporter gene (in a PGL3-based
>vector) in order to test them for inducibility in my system. Unfortunately,
>I've been having a lot of problems getting my subcloning to work. For each
>construct, we've designed two complementary oligos set up so that when they
>anneal, there should be an Nhe I overhang on the upstream end and a Bgl II
>half-site on the downstream end. The plan was to clone these oligos
>directionally into the Bgl II and Nhe I sites in the multiple cloning site.

>Two questions:

>First, does anyone have any hints on what's the best way to anneal two
>complementary oligos prior to a ligation reaction? The oligos range from
>36mers to 80mers.

>Second, in my previous cloning attempts, after cutting the vector with Bgl
>II and Nhe I serially and gel-purifying the fragment, I find that I'm
>getting a lot of self-ligation, as evidenced by lots and lots of colonies
>on my vector-only control plate. At least according to every restriction
>site list I read, Bgl II and Nhe I should not be compatible, but tell that
>to the vector. I've already verified that my enzymes are good by test
>digestions with each enzyme (I can't see the fragment that a double
>digestion should produce because it's only about a dozen nucleotides
>between the sites). Has anyone ever seen this or heard of it before? It
>looks like we're going to have to change our entire subcloning strategy if
>I'm going to get self-ligation between supposedly "incompatible" sites.

>Thanks.

>-- 
>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
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. 

Geetings Tom





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