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Ligation problem: Phosphotasing Sac I ends.

J. Pat Martinez martinez at puccini.CRL.umn.edu
Wed Oct 16 04:47:58 EST 1996

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. 
Any suggestions?

Pat M.

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