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    Dephosphorylation is a common step in traditional cloning workflows to ensure that the vector does not re-circularize during ligation. If a vector is linearized by a single restriction enzyme, or has been cut with two enzymes with compatible ends, use of a phosphatase, such as Shrimp Alkaline Phosphatase (rSAP), to remove the 5' phosphate reduces the occurrence of vector re-closure by intramolecular ligation. Decreased re-circularization reduces the background during subsequent transformation. If the vector is dephosphorylated, it is essential to ensure that the insert contain a 5' phosphate to allow ligation to proceed. Each double-strand break requires that one intact phosphodiester bond be created before transformation (and in vivo repair).

    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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    FAQs for Dephosphorylation

    Protocols for Dephosphorylation