Hsu and Berg, 1978). Prolonged incubation time and high enzyme concentration as well as elevated levels of glycerol and other organic solvents tend to generate star activity (Malyguine Vannier, and Yot, 1980). Maintaining the glycerol concentration to 5% or less is recommended. Since the enzyme is supplied in 50% glycerol, the enzyme added to a reaction should be no more than 10% of the final reaction volume When extra DNA fragments are observed, especially when working with an enzyme for the first time, star activity must be differentiated from partial digestion or contaminating specific endonucleases. first, check to make sure that the reaction condi- tions are well within the optimal range for the enzyme. Then, repeat the digest in parallel reactions, one with twice the activity and one with half the activity of the initial digest. Partial digestion is indicated as the cause when the number of bands is reduced to that expected after repeating the digestion with additional enzyme (or extending incubation time). If extra bands are still evident, contact the supplier's technical support resource for advice. Generally speaking, star activity and contaminating activ ities are more difficult to differentiate Mapping and sequencing he respective cleave es is the best method to distinguish star activity from a partial digest or contaminant activity Site Preference The rate of cleavage at each site within a given DNA substrate can vary(Thomas and Davis, 1975). Fragments containing a subset of sites that are cleaved more slowly than others can result in partial digests containing lighter bands visualized on an ethidium tained agarose gel. Certain enzymes such as Eco Rll require an activator site to allow cleavage(Kruger et al., 1988 ). Substrates lacking the additional site will be cleaved very slowly. For certain enzymes(Nael), adding oligonucleotides containing the site or adding another substrate containing multiple sites can improve utting. In the case of PaeR7l, it has been shown that the sur- rounding sequence can have a profound effect on the cleavage rate(Gingeras and Brooks, 1983). In most cases this rate differ ence is taken in to account because the unit is defined at a point of complete digestion on a standard substrate DNA(e.g, lambda DNA)that contains multiple sites. Problems can arise when certain sites are far more resistant than others, or when highly resistant sites are encountered on substrates other than the stan dard substrate DNA. If a highly resistant site is present in a common cloning vector, then a warning should be noted on the data card or in the catalog Robinson et al(Hsu and Berg, 1978). Prolonged incubation time and high enzyme concentration as well as elevated levels of glycerol and other organic solvents tend to generate star activity (Malyguine, Vannier, and Yot, 1980). Maintaining the glycerol concentration to 5% or less is recommended. Since the enzyme is supplied in 50% glycerol, the enzyme added to a reaction should be no more than 10% of the final reaction volume. When extra DNA fragments are observed, especially when working with an enzyme for the first time, star activity must be differentiated from partial digestion or contaminating specific endonucleases. First, check to make sure that the reaction condi￾tions are well within the optimal range for the enzyme. Then, repeat the digest in parallel reactions, one with twice the activity and one with half the activity of the initial digest. Partial digestion is indicated as the cause when the number of bands is reduced to that expected after repeating the digestion with additional enzyme (or extending incubation time). If extra bands are still evident, contact the supplier’s technical support resource for advice. Generally speaking, star activity and contaminating activ￾ities are more difficult to differentiate. Mapping and sequencing the respective cleavage sites is the best method to distinguish star activity from a partial digest or contaminant activity. Site Preference The rate of cleavage at each site within a given DNA substrate can vary (Thomas and Davis, 1975). Fragments containing a subset of sites that are cleaved more slowly than others can result in partial digests containing lighter bands visualized on an ethidium stained agarose gel. Certain enzymes such as EcoRII require an activator site to allow cleavage (Kruger et al., 1988). Substrates lacking the additional site will be cleaved very slowly. For certain enzymes (NaeI), adding oligonucleotides containing the site or adding another substrate containing multiple sites can improve cutting. In the case of PaeR7I, it has been shown that the sur￾rounding sequence can have a profound effect on the cleavage rate (Gingeras and Brooks, 1983). In most cases this rate differ￾ence is taken in to account because the unit is defined at a point of complete digestion on a standard substrate DNA (e.g., lambda DNA) that contains multiple sites. Problems can arise when certain sites are far more resistant than others, or when highly resistant sites are encountered on substrates other than the stan￾dard substrate DNA. If a highly resistant site is present in a common cloning vector, then a warning should be noted on the data card or in the catalog. 230 Robinson et al