DNA Modification

DNA Dephosphorylation

Digested DNA typically possesses a 5’ phosphate group that is required for ligation. In order to prevent self-ligation, the 5' phosphate can be removed prior to ligation. Dephosphorylation of the 5’ end prohibits self-ligation, enabling the researcher to manipulate the DNA as desired before re-ligating. Generally, it is a good idea to dephosphorylate the linearized vector to facilitate ligation of recombinant DNA molecules.  In fact, vector self-ligation increases the background activity of the cloning process. Dephosphorylation can be accomplished using any of a number of phosphatases, including the Quick Dephosphorylation Kit (NEB #M0508), Shrimp Alkaline Phosphatase (rSAP) (NEB #M0371), Calf Intestinal Alkaline Phosphatase (CIP) (NEB #M0290) and Antarctic Phosphatase (NEB #M0289).

  1. The Mechanism of Dephosphorylation

    Dephosphorylation is the process by which phosphate groups are removed from a molecule by a phosphatase. Removal of phosphate groups from a DNA fragment can prevent ligation. Learn more about dephosphorylation and phosphatases.

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Protocols for DNA Dephosphorylation

Common Applications of Exonucleases and Endonucleases

NEB provides a list of common applications for our exonucleases and endonucleases.

Properties of Exonucleases and Endonucleases

NEB supplies many nucleases; several characteristics should be considered when choosing the one best suited to your particular research needs.