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  • Cloning and Mapping

    DNA Dephosphorylation

    Free DNA typically possesses a 5’ phosphate group, required for ligation. When performing molecular biology techniques, it is not always advantageous to allow the spontaneous self-ligation of DNA ends. Dephosphorylation of the 5’ end prohibits self-ligation, enabling the researcher to manipulate the DNA as desired before re-ligating. For example, when preparing DNA for ligation into a cloning vector, the property of vector self-ligation can be counterproductive. Generally, it is a good idea to dephosphorylate either DNA fragments or the vector to facilitate ligation of recombinant DNA molecules. Dephosphorylation can be accomplished using any of a number of phosphatases, including Shrimp Alkaline Phosphatase (rSAP) (NEB #M0371), Alkaline Phosphatase, Calf Intestinal (NEB #M0290) and Antarctic Phosphatase (NEB #M0289).

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