blunt ligation

Ashok Aiyar aiyar at
Sun Dec 8 12:24:12 EST 1996

On Sun, 08 Dec 1996 16:44:11 -0800, Chang,jihun <j6544 at> 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 Aiyar, Ph.D.
McArdle Laboratory for Cancer Research
University of Wisconsin-Madison

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