Subcloning oligos into expression vectors

Randy Willis willis at gandalf.psf.sickkids.on.ca
Mon Jan 20 09:22:09 EST 1997


David H. Gorski wrote:
> 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.

I've always found that I can just boil the two oligos together and let
them slowly cool to room temperature.  So far, this has worked in our
group for oligos of up to 40nt.


> Second, in my previous cloning attempts, after cutting the vector with BglII 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. 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.


I've found that no matter how efficient the enzymes are supposed to be,
there is always a percentage of vector that is only cut once and that
these will be more likely to ligate and give you colonies.  What we've
taken to doing is,
i) cut with one enzyme first (prob. NheI), gel purify the linearized
fragment, and then cut with the second enzyme, and/or
ii) treat the cleaved vector with calf intestinal alkaline phosphatase
(CIAP) or some such beastie such that the incidence of vector religating
should drop precipitously.

Good luck,

Randall C Willis, Researcher
Biochemistry, Hosp for Sick Children
3522-555 University Ave.
Toronto, ON
M5G 1X8  CANADA

416-813-5933 (ph)  -5022 (fax)
willis at gandalf.psf.sickkids.on.ca



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