Subcloning oligos into expression vectors

David H. Gorski d-gorski at uchicago.edu
Fri Jan 17 21:26:14 EST 1997


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



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