HELP! 3-way ligation

Sean Sweeney ss2 at mole.bio.cam.ac.uk
Fri Apr 19 09:58:46 EST 1996


In article <mcc9-1804961633510001 at 128.84.203.78>, mcc9 at cornell.edu (Maria
C. Costanzo) wrote:

> We're having trouble doing a 3-way ligation in which the vector has PstI
> and BamHI ends, one insert piece has BamHI and NcoI ends, and the other
> insert piece has BspHI (compatible with NcoI) and PstI ends.  Does anyone
> have any tips on strategies for 3-way ligations in general or any info
> about these particular sticky ends?
> Thanks in advance,
> Maria
> 


I have three-way ligations working routinely, again with three different
enzymes. The only difference I have found is with the ligation buffer. I
haven't done any controls but my ligations didn't work in three-ways when 
I used Promega's ligase and buffer but I always have good results with
Gibco/BRL ligase and buffer. Gibco's buffer seems to be pretty heavy with
the polyethylene glycol and this doesn't seem to be the case with
Promega's buffer.

hope this helps

Sean Sweeney
Dept of Genetics
University of Cambridge

ss2 at mole.bio.cam.ac.uk

P.S. I have no connection to either company.



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