Ligation of alkaline phosphatase treated DNA

Dietmar Baas uc05 at rzstud1.rz.uni-karlsruhe.de
Sun Feb 26 18:24:58 EST 1995


Dear Netters,

I have a theoretical problem with the ligation of DNA, treated with
alkaline phosphatase (a.p.) and here is my question:
A.p. is used to prevent ligation of multiple inserts in one vector by 
dephosphorylation of the 5'terminis of the insert. But all books say,
T4 DNA Ligase needs 5'phosphate and 3'OH. This results in a gap in one
of the two strands. How is this gap closed? ATP is present in the 
ligation mixture but I never heared something about kinase activity 
of T4 DNA Ligase.
Another possibility is the "in vivo repair" of the gap after transformation
by enzymes of E.coli (but this sounds like a strange theory).

Has anybody in the net the right answer for this problem?

Ciao, 
      Dietmar
  
  



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