Demonstration of the Principles of Restriction Endonuclease Cleavage Reactions Using Thermostable Bfl I from Anoxybacillus flavithermus

Abstract

Restriction enzymes are basic tools in recombinant DNA technology. To shape the molecular biology experiments, the students must know how to work with these molecular scissors. Here, we describe an integrated set of experiments, introduced in the "Advances in Molecular Biology and Biotechnology" postgraduate course, which covers the important aspects about restriction endonucleases: preparation of cell-lysate, one-column partial-purification, assay, effect of temperature, buffers, Mg2+ ions and glycerol on activity, and quality control of two-column partially purified enzyme. Examples of study questions, which help students understand the theoretical basis of the experiments, have been given.