WHAT ARE THE GENERAL PROPERTIES OF RESTRICTION ENDONUCLEASES? In general, commercial preparations of restriction endonucle- ases are purified and stored under conditions that ensure optimal nd stability over time; namely -20oC. They are com monly supplied in a solution containing 50% glycerol, Tris buffer, EDTA, salt, and reducing agent. This solution will conveniently remain in liquid form at -20C but will freeze at temperatures below -30%C. Those enzymes shipped on dry ice, or stored at -70oC, will have a white crystalline appearance; they revert to a clear solution as the temperature approaches -20C. As a rule repeated freeze-thaw cycles are not recommended for enzyme solutions because of the possible adverse effects of shearing(more on the question, How Stable are Restriction Enzymes? appears As a group(and by definition), Class II restriction endonucle ases require magnesium(Mg)as a cofactor in order to cleave DNA at their respective recognition sites. Most restriction enzymes are incubated at 37C, but many require higher or lower (i. e, Smal requires incubation at 25C)temperatures. Percent activity tables of thermophilic enzymes incubated at 37 C can be found in some suppliers'catalogs. For most reactions, the pl optima is between 7 and 8 and the NaCl concentration betwee 50 and 100 mM. Concentrated reaction buffers for each enzyme are provided by suppliers. Typically each enzyme is profiled for optimal activity as a function of reaction temperature, pH(buffer ing systems), and salt concentration. Some enzymes are also evaluated in reactions containing additional components(BSA detergents). Generally, these characteristics are documented in the published literature and referenced by suppliers Interestingly, a number of commonly used enzymes can dis broad range of stability and performance characteristics under fairly common reaction conditions. They may vary considerably i activity and may exhibit sensitivity to particular components. In effort to minimize these undesirable effects, suppliers often adjust enzyme buffer components and concentrations to ensure optimal performance for the most common applications. There is a wealth of information about the properties of these enzymes in most suppliers'catalogs, as well as on their Web sites. The documentation supplied with the restriction endonuclease should contain detailed information about the enzymes pro- perties and functional purity. It is important to read the Certifi cate of Analysis when using a restriction enzyme for the first Robinson et alWHAT ARE THE GENERAL PROPERTIES OF RESTRICTION ENDONUCLEASES? In general, commercial preparations of restriction endonucle￾ases are purified and stored under conditions that ensure optimal reactivity and stability over time; namely -20°C. They are com￾monly supplied in a solution containing 50% glycerol, Tris buffer, EDTA, salt, and reducing agent. This solution will conveniently remain in liquid form at -20°C but will freeze at temperatures below -30°C. Those enzymes shipped on dry ice, or stored at -70°C, will have a white crystalline appearance; they revert to a clear solution as the temperature approaches -20°C. As a rule repeated freeze-thaw cycles are not recommended for enzyme solutions because of the possible adverse effects of shearing (more on the question, How Stable are Restriction Enzymes? appears below). As a group (and by definition), Class II restriction endonucle￾ases require magnesium (Mg2+ ) as a cofactor in order to cleave DNA at their respective recognition sites. Most restriction enzymes are incubated at 37°C, but many require higher or lower (i.e., SmaI requires incubation at 25°C) temperatures. Percent activity tables of thermophilic enzymes incubated at 37°C can be found in some suppliers’ catalogs. For most reactions, the pH optima is between 7 and 8 and the NaCl concentration between 50 and 100 mM. Concentrated reaction buffers for each enzyme are provided by suppliers. Typically each enzyme is profiled for optimal activity as a function of reaction temperature, pH (buffer￾ing systems), and salt concentration. Some enzymes are also evaluated in reactions containing additional components (BSA, detergents). Generally, these characteristics are documented in the published literature and referenced by suppliers. Interestingly, a number of commonly used enzymes can display a broad range of stability and performance characteristics under fairly common reaction conditions. They may vary considerably in activity and may exhibit sensitivity to particular components. In an effort to minimize these undesirable effects, suppliers often adjust enzyme buffer components and concentrations to ensure optimal performance for the most common applications. There is a wealth of information about the properties of these enzymes in most suppliers’ catalogs, as well as on their Web sites. The documentation supplied with the restriction endonuclease should contain detailed information about the enzyme’s pro￾perties and functional purity. It is important to read the Certifi- cate of Analysis when using a restriction enzyme for the first 232 Robinson et al