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-   -   Why treat plasmid DNA with alkaline phosphatase when making a recombinant plasmid? (http://www.molecularstation.com/forum/dna-techniques/69722-why-treat-plasmid-dna-alkaline-phosphatase-when-making-recombinant-plasmid.html)

Luke . 07-22-2009 03:16 AM

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

Enne815 07-23-2009 09:15 PM

Re: Why treat plasmid DNA with alkaline phosphatase when making a recombinant plasmid
 
Here is my understanding of phosphatases:

Vectors are normally treated with alkaline phosphatases, such as CIP, following a digestion in order to prevent re-ligation of the vector in the absence of the insert. The phosphatase removes the 5' phosphate group so that it cannot interact with the 3' hydroxyl group. Thus, when a ligation is performed you increase your likelihood that your vector contains your insert and not just an empty vector.

I hope it helps!

ajithnandanam 03-28-2013 06:44 AM

Re: Why treat plasmid DNA with alkaline phosphatase when making a recombinant plasmid
 
phosphatase is used to remove phosphate group from the 5'end to prevent self ligation.


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