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

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

Why treat plasmid DNA with alkaline phosphatase when making a recombinant plasmid? - Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.


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Old 07-22-2009, 03:16 AM
Pipette Filler
 
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Default 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.
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Old 07-23-2009, 09:15 PM
Pipette Filler
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Default 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!
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Old 03-28-2013, 06:44 AM
Pipette Filler
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Default 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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