It has been demonstrated that under extreme non-standard conditions, restriction endonucleases are capable of cleaving sequences which are similar but not identical to their defined recognition sequence. This altered or relaxed specificity has been termed ""star"" activity. It has been suggested that star activity may be a general property of restriction endonucleases and that any restriction endonuclease can be made to cleave noncanonical sites under certain extreme conditions. The manner in which an enzyme's specificity is altered depends on the enzyme and on the conditions employed to induce the star activity. The most common types of altered activity are single base substitutions, truncation of the outer bases in the recognition sequence, and single-strand nicking. Star activity is completely controllable in the vast majority of cases and is generally not a concern when performing restriction endonuclease digests. New England Biolabs' enzymes will not exhibit star activity when used under recommended conditions in their supplied NEBuffers. Listed below are reaction conditions known to induce or inhibit star activity. Conditions that Contribute to Star Activity 1. High glycerol concentration [> 5% v/v] 2. High units to µg of DNA ratio [Varies with each enzyme, usually >100 units/µg] 3. Low ionic strength [< 25 mM] 4. High pH [> pH 8.0] 5. Presence of organic solvents [DMSO, ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, sulphalane] 6. Substitution of Mg++ with other divalent cations [Mn++, Cu++, Co++, Zn++] Inhibiting Star Activity If you are concerned about star activity, we recommend the following guidelines. 1. Use as few units as possible to get a complete digestion. This avoids overdigestion and reduces the final glycerol concentration in the reaction. 2. Make sure the reaction is free of any organic solvents such as alcohols which might be present in the DNA preparation. 3. Raise the ionic strength of the reaction buffer to 100-150 mM (provided the enzyme is not inhibited by high salt). 4. Lower the pH of the reaction buffer to pH 7.0. 5. Use Mg++ as the divalent cation.