Benzonase is a genetically engineered endonuclease from Serratia marcescens (1, 2). Theenzyme is produced and purified from E. coli strain W3110, a mutant of strain K12, containing theproprietary pNUC1 production plasmid (3, 4). Structurally, the protein is a dimer of identical 245amino acid, ~30 kDa subunits with two essential disulfide bonds (5–8). This promiscuousendonuclease attacks and degrades all forms of DNA and RNA (single stranded, double stranded,linear and circular) and is effective over a wide range of operating conditions (9). The enzymecompletely digests nucleic acids to 5′-monophosphate terminated oligonucleotides 2–5 bases inlength (2, 10). Although the nuclease is capable of cleavage at nearly all positions along a nucleicacid chain, sequence-dependent preferences have been demonstrated (11). The enzyme prefersGC-rich regions in dsDNA while avoiding d(A)/d(T)-tracts. Benzonase is now available fromNovagen in two grades, Purity > 99% and Purity > 90%. Both preparations possess exceptionallyhigh specific activities and are supplied free from measurable protease activities and viralcontaminants. Benzonase is ideal for a wide variety of applications where complete digestion ofnucleic acids is desirable.Unit definitionOne unit of Benzonase Nuclease is defined as the amount of enzyme that causes a ∆A260 of 1.0 in30 min, which corresponds to complete digestion of 37 μg of DNA. Standard reaction conditionsare 1 mg/ml sonicated DNA substrate in 50 mM Tris-HCl pH 8.0, 0.1 mg/ml BSA, 1 mM MgCl2,incubated at 37°C; measured as perchloric acid-soluble digestion product.StorageBenzonase Nuclease is supplied in 50% glycerol containing 50 mM Tris-HCl pH 8.0,20 mM NaCland 2 mM MgCl2. The enzyme preparation is stable for 2 years when stored at –20°C.DO NOT store at –70°C as freezing Benzonase Nuclease results in loss of activity.