TA cloning of a PCR product generate by Vent DNA polymerase

Andrey S. Shaw shaw at immunology.wustl.edu
Sat Aug 2 22:29:11 EST 1997


Yes and No.  We can get  a plasmid amplified with Vent to ligate to
itself, but theoritically this should not be  as the ends (which come
from the oligos) are not phosphorylated.  Thus, we routinely add  T4
kinase to our ligation reactions first and then after 30 minutes add
ligase and cool to 14 degrees.  You willl find that the kinase and
ligase buffer are identical and the ATP in the ligase buffer is OK for
the kinase. 

andrey shaw



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