Star activity is of concern if extra banding can cause misinterpretation of results in genotyping and mutational analysis procedures. Experimental design can promote star activity. Small reaction volumes are more likely to contain glycerol concentrations of 5% or greater, a condition known to increase star activity. A 5% glycerol concentration occurs when setting up a double digest in a 20 µl reaction using 1 µl of each enzyme. Overnight digests are more likely to generate star activity. For tips on avoiding star activity, please click here.