Antarctic Phosphatase has a strict requirement for Zinc. It is active in all restriction enzyme NEBuffers 1.1 , 2.1 , 3.1 and CutSmart® Buffer as well as NEBuffers 1-4 and the unique NEBuffer EcoRI only when supplemented with 10X Antarctic Phosphatase Buffer to a final concentration of 1X. This buffer provides the zinc required for Antarctic Phosphatase activity. Antarctic Phosphatase can then be added directly to the digested DNA. Heat inactivation of the restriction enzyme before dephosphorylation reaction is not necessary. To see its % functional activity in Cutsmart, and that of other DNA modifying enzymes in the cloning workflow, refer to the Activity of DNA Modifying Enzymes in CutSmart® Buffer chart.