blunt ligation

Ashok Aiyar aiyar at ebv.oncology.wisc.edu
Sun Dec 8 12:24:12 EST 1996


On Sun, 08 Dec 1996 16:44:11 -0800, Chang,jihun <j6544 at unitel.co.kr> wrote:
>Dear bionetter.
> 
>I'm trying to ligate Hind III-digested genomic DNA into SmaI-digested 
>pBluescript SK vector.
>but, final white transformants I got were so few.
>I want many white colonies.
>Please, give me your help.

Have you blunted your HindIII digested genomic DNA?  If you have done
so, you may wish to reduce the concentration of ATP in the ligation.  I
have had excellent success with blunt ligations of large DNA molecules
using a final ATP concentration of 50 micromolar.

Finally, since your HindIII digested fragments (originally) have "sticky"
ends, why not simply ligate them into HindIII digested pBlueScript?

Ashok
-- 
Ashok Aiyar, Ph.D.
McArdle Laboratory for Cancer Research
University of Wisconsin-Madison



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