Help with a knotty ligation?
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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