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. 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, 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.
