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Home Q & A Board Biology-Related Homework Help Biochemistry
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extraelle extraelle
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10 years ago
When attempting to create a recombinant DNA molecule, by inserting a foreign sequence into the vector plasmid; what is the point (and how does it achieve it) of treating the plasmid DNA with alkaline phosphatase?

I think it has something to do with the orientation of the inserted sequence, but unsure how the treatment achieves this.
thats right, it makes sense now
thanks guys
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#1
10 years ago
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.
wrote...
#2
10 years ago
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.
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