simont at post.its.mcw.edu
Wed Feb 21 10:57:02 EST 1996
We have also had similar ligation problems and have found that in our
case, the problem was resolved by checking the ligation buffer, rather
than changing DNA purification techniques. We made some fresh ligation
buffer by using Pharmacia One Phor All buffer plus the appropriate
amount of ATP and everything worked like you know it ought to!
It may well be that repeated freeze thawing of the 10x ligation buffer
provided with the ligase causes/accelerates the breakdown of the ATP in
the ligase buffer, leading to poor ligation results. Certainly the
documentation for the ligase we use from Promega suggests that you
aliquot the 10x ligase buffer when you receive it to avoid repeated
If you havent already checked this, it might be worth a look.
Good luck with the ligations!
Medical College of Wisconsin, Milwaukee.
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