time, as it may provide important information concerning partic ular substrate DNAs or alternative reaction conditions for a spe cific application What Insight Is Provided by a Restriction Enzyme's Quality Control Data? Restriction enzymes are isolated from bacterial strains that contain a variety of other enzyme activities required for normal cell function. These additional activities include other nucleases phosphatases, and polymerases as well as other dna binding pro- teins that may inhibit restriction enzyme activity In preparations where trace amounts of these activities remain the end-structure of the resulting DNA fragments may be degraded, thus inhibiting subsequent ligation. Likewise plasmid substrates may be nicked thus reducing transformation efficiencies Ideally the restriction enzyme preparation should be purified to homogeneity and free of any detectable activities that might inter fere with digestion or inhibit subsequent reactions planned for the resulting DNA fragments. In order to provide researchers with a practical means to conveniently evaluate the suitability of a given restriction enzyme preparation, suppliers include a Certificate of Analysis with each product, detailing the preparation,s per formance in a defined set of Quality Control Assays. In order to establish a standard reference for the amount of enzyme and sub- strate used in these assays, each supplier must first define the unit substrate and reaction conditions for each product Unit Definition a unit of restriction endonuclease is defined as the amount of zyme required to completely cleave l ug of substrate DNA suS- pended in 50 ul of the recommended reaction buffer in one hour at the recommended assay buffer and temperature. The DNA most often used is bacteriophage Lambda or another well characterized substrate. Note that the unit definition is not based on classic enzyme kinetics. The enzyme molar concentration is in excess. A complete digest is determined by the visualized pattern of cleaved DNA fragments resolved by electrophoresis on an ethidium bromide-stained gel. Some restriction enzymes will behave differently when used outside the parameters of the unit definition. The number of sites(site density)or the particular type of DNA substrate may have an effect on"unit activity, " but it is not always proportional(Fuchs and Blakesley, 1983) Restriction Endonucleases 233time, as it may provide important information concerning partic￾ular substrate DNAs or alternative reaction conditions for a spe￾cific application. What Insight Is Provided by a Restriction Enzyme’s Quality Control Data? Restriction enzymes are isolated from bacterial strains that contain a variety of other enzyme activities required for normal cell function. These additional activities include other nucleases, phosphatases, and polymerases as well as other DNA binding pro￾teins that may inhibit restriction enzyme activity. In preparations where trace amounts of these activities remain, the end-structure of the resulting DNA fragments may be degraded, thus inhibiting subsequent ligation. Likewise plasmid substrates may be nicked, thus reducing transformation efficiencies. Ideally the restriction enzyme preparation should be purified to homogeneity and free of any detectable activities that might inter￾fere with digestion or inhibit subsequent reactions planned for the resulting DNA fragments. In order to provide researchers with a practical means to conveniently evaluate the suitability of a given restriction enzyme preparation, suppliers include a Certificate of Analysis with each product, detailing the preparation’s per￾formance in a defined set of Quality Control Assays. In order to establish a standard reference for the amount of enzyme and sub￾strate used in these assays, each supplier must first define the unit substrate and reaction conditions for each product. Unit Definition A unit of restriction endonuclease is defined as the amount of enzyme required to completely cleave 1mg of substrate DNA sus￾pended in 50ml of the recommended reaction buffer in one hour at the recommended assay buffer and temperature. The DNA most often used is bacteriophage Lambda or another well￾characterized substrate. Note that the unit definition is not based on classic enzyme kinetics. The enzyme molar concentration is in excess. A complete digest is determined by the visualized pattern of cleaved DNA fragments resolved by electrophoresis on an ethidium bromide-stained gel. Some restriction enzymes will behave differently when used outside the parameters of the unit definition. The number of sites (site density) or the particular type of DNA substrate may have an effect on “unit activity,” but it is not always proportional (Fuchs and Blakesley, 1983). Restriction Endonucleases 233