Abstract
Mapping the precise position of endonucleolytic cleavage sites is a fundamental experimental technique used to describe the function of a homing endonuclease. However, these proteins are often recalcitrant to cloning and over-expression in biological systems because of toxicity induced by spurious DNA cleavage events. In this chapter we outline the steps to successfully express a homing endonuclease in vitro and use this product in nucleotide-resolution cleavage assays.