The quality and cleanliness of the DNA are major factors that can affect enzyme activity. Restriction Enzymes active in CutSmart™ Buffer, but not as active in NEBuffer 2.1 and/or 3.1 (our higher salt buffers), can be inhibited by salt in the reaction. DNA purification procedures that use spin columns can result in DNA solutions with significant levels of salt that can carry over into the reaction and inhibit enzyme activity. To prevent this, we recommend that the DNA solution added to the reaction be no more than 25% of the total reaction volume. This can be attained by adjusting the total reaction volume accordingly.