I'm having difficulty cloning a 7 kb Sac I fragment into pUC 19. One
problem has been the phosphotasing of pUC 19/SacI. I phosphatased the
vector twice with shrimp alkaline phosphotase. Comparing transformations
of vector without ligase and vector with ligase, I get thousands of blue
colonies for the vector with ligase. Self ligation of the vector in the
absence of insert has also been confirmed by gel electrophoresis.
Apparently the 5' *recessed* phosphates are not being removed completely.