What Can I Do to Prevent Star Activity in my Restriction Enzyme Digestions?

Any restriction endonuclease can be subject to star activity. Generally, star activity (i.e., the enzyme cuts a sequence that is an imperfect match to its known target) is caused by nonoptimal reaction conditions. ⁽¹⁾, ⁽²⁾ These include:

• High concentration of glycerol (>5% v/v).
• High ratio of enzyme units to micrograms of DNA. This ratio depends on each enzyme but generally means >10 units per microgram DNA.
• Low ionic strength.
• High or low pH.
• Presence of organic solvents such as DMSO, ethanol, ethylene glycol, dimethylacetamide, dimethylformamide or sulphalane.
• Substitution of other divalent cations [e.g., Mn²⁺, Cu²⁺, Co²⁺ or Zn²⁺] for Mg²⁺.

To prevent star activity, we recommend the following guidelines:

• Use as few units of restriction enzyme as possible for a complete digestion, which avoids overdigestion of the DNA and reduces the final glycerol concentration in the reaction. We recommend using one unit of restriction enzyme for every microgram of DNA and incubating the reaction for 1 hour. Note: Supercoiled DNA requires three- to fivefold more enzyme than linear DNA.
• Make sure the reaction is free of any organic solvents such as alcohols, which may have carried over during DNA purification.
• Increase the ionic strength of the reaction buffer to 50–150mM as long as the enzyme is not inhibited by high salt.
• Keep the pH in the range of 7.2–8.5, depending on the restriction endonuclease.
• Use only Mg²⁺ as the divalent cation in the reaction buffer.