If You Could Select among Several Restriction Enzymes for Your Application, What Criteria Should You Consider to Make the Most Appropriate Choice? Each restriction endonuclease is a unique enzyme with individ- ual characteristics, which are usually listed in suppliers'catalogs and package inserts. When using an unfamiliar enzyme, these data should be carefully reviewed. In addition some enzymes provide additional activities that may impact the immediate or down- stream application. Ease of Use For many applications it is desirable and convenient to use 1ul per reaction. Most suppliers offer standard enzyme concentrations ranging from 2000 to 20,000 units/ml(2-20 units/ul). In addition many suppliers also offer these enzymes in high concentration (often up to 100,000 units/ml), either as a standard product, or through special order. Enzymes sold at 10 to 20 units/ul are common and usually lend themselves for use in a wider variety of applications. When planning to use enzymes available only in lower concentrations(near 2000 units/ml), be sure to take the final glycerol concentration and reaction volume into account. B following the recommended conditions and maintaining the final glycerol concentration below 5%, you can easily avoid star activit ty. Star Activity When subjected to reaction conditions at the extremes of their operating range, restriction endonucleases are capable of cleaving sequences that are similar, but not identical, to their canonical recognition sequences. This altered specificity has been termed "star activity. Star sites are related to the recognition site, usually differing by one or more bases. The propensity for exhibiting star activity varies considerably among restriction endonucleases. For a given enzyme, star activity will be exhibited at the same relative level in each lot produced, whether isolated from a recombinant or a nonrecombinant source Star activity was first reported for EcoRi incubated in a low ionic strength high pH buffer(Polisky et al., 1975). Under these conditions, while this enzyme would cleave at its canonical site (G/AATTC), it also recognized and cleaved at N/AATTC. This reduced specificity should be a consideration when planning to use a restriction endonuclease in a nonoptimal buffer. It was also found that substituting Mn**for Mg+ can result in star activity Restriction EndonucleasesIf You Could Select among Several Restriction Enzymes for Your Application,What Criteria Should You Consider to Make the Most Appropriate Choice? Each restriction endonuclease is a unique enzyme with individ￾ual characteristics, which are usually listed in suppliers’ catalogs and package inserts. When using an unfamiliar enzyme, these data should be carefully reviewed. In addition some enzymes provide additional activities that may impact the immediate or down￾stream application. Ease of Use For many applications it is desirable and convenient to use 1ml per reaction. Most suppliers offer standard enzyme concentrations ranging from 2000 to 20,000 units/ml (2–20 units/ml). In addition many suppliers also offer these enzymes in high concentration (often up to 100,000 units/ml), either as a standard product, or through special order. Enzymes sold at 10 to 20 units/ml are common and usually lend themselves for use in a wider variety of applications. When planning to use enzymes available only in lower concentrations (near 2000 units/ml), be sure to take the final glycerol concentration and reaction volume into account. By following the recommended conditions and maintaining the final glycerol concentration below 5%, you can easily avoid star activity. Star Activity When subjected to reaction conditions at the extremes of their operating range, restriction endonucleases are capable of cleaving sequences that are similar, but not identical, to their canonical recognition sequences. This altered specificity has been termed “star activity.” Star sites are related to the recognition site, usually differing by one or more bases. The propensity for exhibiting star activity varies considerably among restriction endonucleases. For a given enzyme, star activity will be exhibited at the same relative level in each lot produced, whether isolated from a recombinant or a nonrecombinant source. Star activity was first reported for EcoRI incubated in a low ionic strength high pH buffer (Polisky et al., 1975). Under these conditions, while this enzyme would cleave at its canonical site (G/AATTC), it also recognized and cleaved at N/AATTC. This reduced specificity should be a consideration when planning to use a restriction endonuclease in a nonoptimal buffer. It was also found that substituting Mn2+ for Mg2+ can result in star activity Restriction Endonucleases 229