Why treat plasmid DNA with alkaline phosphatase when making a recombinant plasmid?

2 Comments

1. Evan M says:

   November 1, 2009 at 1:52 am

   Primarily, I think the purpose is to avoid self-ligation of the plasmid after the restriction is performed. It seems to me that treatment with AP would only be crucial when using a single restriction enzyme, or when performing a blunt end ligation.

   On the other hand, even when cutting with different enzymes, restriction efficiency is rarely 100%. AP treatment would likely prevent religation of plasmids that have only been cut once.

   I’m not sure that AP treatment would facilitate proper insert orientation.

   Hope this helps.
2. sonnesolaris says:

   November 1, 2009 at 1:52 am

   Also important to note that ligase is more efficient in annealing DNA that have only one phosphate group (between pieces to be ligated). So treating the plasmid with a phosphatase, while leaving the untreated insert with phosphates facilitates successful integration of sequence of interest into vector.