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 Recombinant Albumin (rAlbumin) at a final concentration of 100 μg/ml for optimal activity. When required, rAlbumin is supplied as a 10 mg/ml (100X) stock and should be added to the reaction mixture. Restriction enzymes that do not require albumin for optimal activity are not adversely affected if albumin is present in the reaction.

The buffer must be completely thawed before use. The 10X stock should be used at a final concentration of 1X. For example, a 50 µl reaction should be assembled by first adding Nuclease-free water, then 5 µl of the 10X buffer followed by substrate and enzyme.