FAQ: How should the NEBuffer be used?

New England Biolabs provides a color-coded 10X NEBuffer with each restriction enzyme to ensure (100%) optimal activity. The buffer should be used at 1X concentration in the reaction. Some restriction enzymes require bovine serum albumin (BSA) at a final concentration of 100 μg/ml for optimal activity. When required, BSA is supplied as a 10 mg/ml (100X) stock and should be added to the reaction mixture. Restriction enzymes that do not require BSA for optimal activity are not adversely affected if BSA is present in the reaction.

The buffer must be completely thawed before use. Dilute the 10X stock with dH2O to a final concentration of 1X. Add the water first, buffer next, BSA if required, the DNA solution and finally the enzyme. A typical 50 µl reaction should contain 5 µl of 10X NEBuffer with the rest of the volume coming from the DNA solution, enzyme and dH2O.