Genomic Digests When Preparing Genomic DNA for Southern Blotting, How Can You Determine If Complete Digestion Has Been Obtained? 244 What Are Your Options If You Must Create Additional Rare or Unique Restriction Sites? 247 Troubleshooting∴. 255 What Can Cause a Simple Restriction Digest to Fail? 255 The Volume of Enzyme in the Vial Appears Very Low. Did eakage Occur during Shipment The Enzyme Shipment Sat on the Shipping Dock for Analyzing Transformation Failure and Other Multiple-Step procedures Involving Restriction Enzymes 260 Bibliography .262 BACKGROUND INFORMATION Molecular biologists routinely use restriction enzymes as ke eagents for a variety of applications including genomic mapping, restriction fragment length polymorphism(RFLP)analysis, DNA sequencing, and a host of recombinant DNA methodologies. Few would argue that these enzymes are not indispensable tools for the variety of techniques used in the manipulation of DNA, but like many common tools that are easy to use, they are not always applied as efficiently and effectively as possible. This chapter focuses on the biochemical attributes and requirements of re striction enzymes and delivers strategies to optimize their use in mple and complex reactions Which Restriction Enzymes Are Commercially Available? While as many as six to eight types of restriction endonucleases have been described in the literature Class li restriction endonu cleases are the best known, commercially available and the most useful. These enzymes recognize specific DNA sequences cleave each DNA strand to generate termini with 5 phosphate and 3 hydroxyl groups. For the vast majority of enzymes charac- terized to date within this class, the recognition sequence is nor mally four to eight base pairs in length and palindromic. The point of cleavage is within the recognition sequence. A variation on th theme appears in the case of Class IIS restriction endonucleases. 226 Robinson et alGenomic Digests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244 When Preparing Genomic DNA for Southern Blotting, How Can You Determine If Complete Digestion Has Been Obtained? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244 What Are Your Options If You Must Create Additional Rare or Unique Restriction Sites? . . . . . . . . . . . . . . . . . . . 247 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 What Can Cause a Simple Restriction Digest to Fail? . . . . 255 The Volume of Enzyme in the Vial Appears Very Low. Did Leakage Occur during Shipment? . . . . . . . . . . . . . . . . . . . . 259 The Enzyme Shipment Sat on the Shipping Dock for Two Days. Is It still Active? . . . . . . . . . . . . . . . . . . . . . . . . . 259 Analyzing Transformation Failure and Other Multiple-Step Procedures Involving Restriction Enzymes . . . . . . . . . . . . 260 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262 BACKGROUND INFORMATION Molecular biologists routinely use restriction enzymes as key reagents for a variety of applications including genomic mapping, restriction fragment length polymorphism (RFLP) analysis, DNA sequencing, and a host of recombinant DNA methodologies. Few would argue that these enzymes are not indispensable tools for the variety of techniques used in the manipulation of DNA, but like many common tools that are easy to use, they are not always applied as efficiently and effectively as possible. This chapter focuses on the biochemical attributes and requirements of re￾striction enzymes and delivers strategies to optimize their use in simple and complex reactions. Which Restriction Enzymes Are Commercially Available? While as many as six to eight types of restriction endonucleases have been described in the literature, Class II restriction endonu￾cleases are the best known, commercially available and the most useful. These enzymes recognize specific DNA sequences and cleave each DNA strand to generate termini with 5¢ phosphate and 3¢ hydroxyl groups. For the vast majority of enzymes charac￾terized to date within this class, the recognition sequence is nor￾mally four to eight base pairs in length and palindromic.The point of cleavage is within the recognition sequence. A variation on this theme appears in the case of Class IIS restriction endonucleases. 226 Robinson et al