Some restriction endonucleases generate a single-stranded nick at their recognition sequences in the presence of ethidium bromide (EtBr). This nick can then be extended to a single-stranded gap in which mutations can be introduced by a variety of techniques. To date, the templates used in these studies have largely contained a single recognition site for a given enzyme. Therefore, we have extended these studies to twelve enzymes for which multiple recognition sites exist in the template and show that, under appropriate conditions, one single-stranded nick is introduced per plasmid molecule.