Ligation of alkaline phosphatase treated DNA

kang at msvax.mssm.edu kang at msvax.mssm.edu
Sun Mar 5 22:00:37 EST 1995


In article <3ir2ka$mep at nz12.rz.uni-karlsruhe.de>, uc05 at rzstud1.rz.uni-karlsruhe.de (Dietmar Baas) writes:
>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
>  
>  
Hi

You are right. The gap is filled in vivo by the kination and ligation after
transformation. I addressed the similar question in the issue of Biotechniques
1993, 15(4), 659.

Chulho Kang
Wizard of TM.




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