Help with a knotty ligation?

Tim Fitzwater TFITZWATER at somalogic.COM
Tue Dec 18 17:30:33 EST 2001

1.  Are you dephosphorylating your vector?  A small percentage of vector is
probably single-cut and is able to religate with high efficiency.  Add
Shrimp Alkaline Phosphatase during the restriction of the vector even when
using 2 different restriction sites.
2.  If the vector has a unique restriction site between the BamH I and Nhe I
sites that does not occur in the fragment you are trying to clone, add that
restriction enzyme to the heat-inactivated ligation reaction and incubate
for 20 minutes.  Heat inactivate the restriction enzyme and transform.  This
will also eliminate religated single-cut vector.
3.  If there is a poison sequence, try incubating the bacteria at 30 degrees
rather than 37 degrees.  Sometimes this helps.


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