Blunt end ligations - another question

Klaus.Matthaei at anu.edu.au Klaus.Matthaei at anu.edu.au
Wed Feb 9 22:40:21 EST 1994


>Hi netters,
>
>has anybody tried to do blunt end ligation in the presence of a blunt-cutter?
>
>this should work for blunted DNA fragments that won't restore the original
>site in the vector once ligated. The idea is to push the rxn into the
>fragment ligated direction w/o having to CIP the vector.
>
>->      which enzymes do work?
>
>->      which buffers did you use?
>


Yep it works,  Stratagene use SrfI but at about $US2.50/U it is very XXXy. 
We also use SmaI if your insert doesn't contain a SmaI site.

Hint DO NOT ligete overnight since the enzyme dies but your ligase may not.
 Therefore ligate for about an hour, heat kill and transform.

Cheers, Klaus
(PS, Collaborator with MPIB Freiburg)
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Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei at anu.edu.au

"Think twice - Do once"
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