Blunt end Ligation

Rafa Maldonado Rafael at genetics.med.utah.edu
Wed Mar 16 21:17:27 EST 1994


     I have some ideas, I don't know if they are very stupid, but...

     Perhaps NdeI is cutting inespecifycally due to star activity.
Promega says that NdeI has star activity in digestions with >10%
glycerol. If there is small fragments of plasmid which contain the
replications origin, they will ligate more eficiently, and maybe the
give more transformation frequency than the more abundant 9.1 kb
plasmid, or perhaps they have compatible ends.
     The gel purification of fragments doesn't clean of all the others
fragments in the reaction. I have cut a plasmid in four fragments,
purified the four frgaments separately and all the fragments hybridize
with the other three. So you can carry samll fragments with you big
fragment.
    Other posibility is hte presence of reorganizated plasmid in your
preparation, because the plasmid is unstable or something like that.
When you transform, you get that plasmids because they have no Xho or
Nde sites. You need the carryover in the agarose again...
     Sorry, no more better ideas!



----------------------------------
Rafael Maldonado
Howard Hughes Medical Insitute
University of Utah
Rafael at genetics.med.utah.edu



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