Ligations

Mukund Nori mn5y at krebs.acc.Virginia.EDU
Thu Sep 6 23:15:34 EST 1990


I have been suddenly having the utmost frustrating experiences with ligations.
I am not even able to cut a plasmid and get it to religate to itself.  So far,
I have used BRL's T4 ligase and the 5X buffer that comes with it.  I have
purified the DNA frm agarose gels using GeneClean, Freeze-squeeze and
electroelution into a trough cut downcurrent of the band and following it into
this selfsame trough.  One of my colleagues has even started with triple
banded (on CsCl) material, all to no avail.  Yes we have tried fresh enzyme
and all fresh solutions.  We even used BRL's supercomp cells to try to get
the presumed-ligated stuff in them.  So far, ZILCH!!!  Suggestions and ideas
EXTREMELY welcome.  Thanks in advance.

 ******************************************************************
 ___Raistlin___  			Mukund Nori
 Raistlin at Virginia.EDU			mn5y at krebs.acc.Virginia.EDU
      "VIOLENCE IS THE LAST RESORT OF THE INCOMPETENT" Asimov 



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