Ligation problems

John and Alex pulitzer at plauto.csata.it
Sat Mar 18 12:44:16 EST 1995


bckraev at wawona ("Alexander Kraev" bckraev at aeolus.ethz.ch) wrote:
>

> >
> >There is no real special technique, you just have to accept that it
> is not going to be as efficient as any sticky end-ligation. All you need
> for success is: phosphatased, gel purified vector; a lot of ligase ( 1-5
> Weiss units in 10 ul reaction mixture) and an excess of insert. Typically,
> 20 ng of vector and 20-100 ng of an insert are ligated in 10 ul. With
> average competent cells ( 10E7 colonies per ug ) this reaction should produce 
> a total of 200 colonies, of which at least 30% are recombinant. These
> figures in fact show that such a ligation is about 10E4 times less efficient
> than an average sticky-end one. Hope this helps, 
> 
Thats an impressive calculation! Does it take into account efficiency of  
circularization of the 3kb insert? Anyway I wish I knew how to do it.
Could you let us have some reference or hint on how you calculated the
10E4 factor difference? Thanks.

John Pulitzer
Dept. of Genetics
U. of Naples
Italy
  



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