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    Dephosphorylation

    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).

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