Sticky-blunt ligations

J. Pat Martinez martinez at puccini.CRL.umn.edu
Wed Sep 11 11:17:24 EST 1996


Try using the SmaI neoschizomer, XmaI.   It recognize the exact same 6 bp
as Sma I but leaves sticky ends.  You may also want to make sure to remove
the stuffer fragment generated from the double digestion of the vector. 
You can do this either by  differential PEG precipitation, chromatogrophy,
or gel purification.  

Pat M.

In article <516lck$gn0 at taurus.adnc.com>, noone at imnr.com (reset) wrote:

> Hell all!
> 
> I'm having trouble with a ligation and I am looking for some advice. I
> am attempting to clone an insert into a vector via Sst I and Sma I
> sites in both insert and vector. Sma I is a blunt-cutter, Sst I has a
> 4-base 5' overhang. The ligation doesn't work - transformations have
> yielded low efficiencies, and colonies that come up appear to be from
> a contaminating fragment from the gel isolation of the insert - i.e.
> the original vector containing the isolated insert. 
> 
> Any ideas about what is happening, and any ideas to solve the problem
> would be appreciated.
> 
> Thanks!
> 
> Jose E. N. Gonzales
> The Immune Response Corporation



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