Preface (2004)

Although mastery of the techniques in this manual will enable the reader to pursue research in molecular genetics, the manual is not intended to be a substitute for graduate-level courses in molecular biology or a comprehensive textbook in the field. Introductory texts that we recommend include: Molecular Biology of the Gene (4th ed.), by J.D. Watson, N.H. Hopkins, J.W. Roberts, J.A. Steitz, and A.M. Weiner; Molecular Genetics: An Introductory Narrative, by G.S. Stent and R. Calendar; From Genes to Clones, by E.-L. Winnacker; and Genetics and Molecular Biology, by R. Schleif. In addition, An Introduction to Genetic Analysis, by D.T. Suzuki, A.J.F. Griffiths, J.H. Miller, and R.C. Lewontin, is a good place to learn classical genetics, and The Molecular Biology of the Cell, by B. Alberts, D. Bray, J. Lewis, M. Raff, K. Roberts, and J.D. Watson is a compendium of useful information about many aspects of cellular and molecular biology. Finally, we strongly recommend that readers gain first-hand experience in basic techniques and safety procedures by working in a molecular biology laboratory. An inevitable hazard of manual writing is that protocols become obsolete or that new techniques are developed—usually just as the pages are going to press. This problem is particularly severe in a fast-moving field such as molecular genetics. To safeguard our manual from inexorable obsolescence, and as a means of correcting errors in a timely manner, we provide a quarterly updating service. The looseleaf format facilitates the replacement of pages that contain errors or have become obsolete and will accommodate the addition of new protocols to the appropriate section of the manual. The publisher can provide further details about subscribing to the quarterly supplements. This publication is available in looseleaf, CD-ROM and online formats. The material covered in each version is identical. For looseleaf purchasers, four binders are provided to accommodate the growth of the manual via the quarterly update service. The looseleaf format of the binders allows easy insertion of new pages, units, and chapters that are added. The index and table of contents are updated with each supplement. CD-ROM purchasers receive a completely new disc containing the entire manual every quarter. The CD-ROM User's Guide describes in detail the many features designed for accessing information presented in the CD. Topics of interest can be located primarily by using either the search index (by typing in the desired word or string of words) or by scanning through the expandable table of contents and selecting the desired units. In addition, hyperlinks jump between related units when cross-references are selected and from solutions and reagents listed in the materials list to their corresponding recipes. Subjects in this manual are organized by chapters and sections, and protocols are contained in units. Units generally describe a method and include one or more protocols with listings of materials, the protocol steps and annotations, recipes for unique reagents and solutions, and commentaries on the “hows” and “whys” of the method. Overview units contain theoretical discussions that lay the foundation for subsequent protocols. Page numbering in the looseleaf version reflects this modular arrangement; for example, page 15.1.3 refers to Chapter 15 (PCR), Unit 1 (standard procedures and optimization), page 3 of that particular unit. Many reagents and procedures are employed repeatedly throughout the manual. Rather than duplicate this information, cross-references among units are used extensively. Early chapters (and appendices) describe these commonly used techniques (basic microbiology and basic manipulations of enzymes, DNA, and RNA), while later chapters describe their application (constructing libraries, DNA sequencing, mutagenesis, transfection, and protein analysis). Thus, whenever a particular enzyme is used in a protocol, the appropriate unit in Chapter 3—describing reaction conditions for that enzyme—is cross-referenced (e.g., UNIT Unavailable for reverse transcriptase). Similarly, throughout the book readers are referred to UNIT Unavailable for spreading or streaking a plate, to UNIT Unavailable for phenol extraction/alcohol precipitation, to UNIT Unavailable for agarose gel electrophoresis, and so on. By turning to these units, the reader will find instructions for the techniques, recipes for relevant reagents, and commentary. As a result, protocols in the later chapters of the book—which can be lengthy and complex—are not overburdened with steps describing auxiliary procedures required to prepare, purify, and analyze the sample or molecule of interest. Because this publication is first and foremost a compilation of molecular biology techniques we have not provided extensive instructive material. We have, however, included explanatory information where required to help readers gain an intuitive grasp of the procedures. First, the chapter, section, and unit introductions describe how the protocols that follow connect to each other. Second, annotations to the actual protocol steps describe what is happening. Third, the commentary sections that close each unit give background information and review the relevant literature so that readers are aware of the historical and theoretical development of a technique, as well as alternative approaches. Fourth, key references are sometimes included to inform users of particularly useful background reading or original descriptions or applications of the technique. Finally, in some cases special “overview” units have been included early in the chapter. Many units in the manual contain groups of protocols. The basic protocol is presented first in each unit and is generally the recommended approach. Alternate protocols are provided where different equipment or reagents can be employed to achieve similar ends, where the starting material requires a variation in approach, or where requirements for the end product differ from those in the basic protocol.Support protocols describe additional steps that are required to perform the basic or alternate protocols; these steps are separated from the core protocol because they might be applicable to other uses in the manual, or because they are performed in a time frame separately from the basic protocol steps. Reagents required for a protocol are listed in the “materials” list before the procedure begins. Many are common stock solutions, others are commonly used buffers or media, while others are solutions unique to a particular protocol. Recipes for the latter solutions are provided in each unit, following the protocols (and before the commentary) under the heading Reagents and Solutions. It is important to note that the names of some of these special solutions might be similar from unit to unit (e.g., hybridization solution, high-salt solution, etc.) while the recipes differ; thus, make certain that reagents are prepared from the proper recipes. On the other hand, recipes for commonly used buffers and media are provided once in an appendix or in early units in the manual. These universal recipes are cross-referenced parenthetically in the materials lists rather than repeated with every usage. NOTE: Deionized, distilled water should be used in all protocols in this manual, and in the preparation of all reagents and solutions. In some instances throughout the manual, we have recommended commercial suppliers of chemicals, biological materials, kits, or equipment. This has been avoided wherever possible, because preference for a specific brand is subjective and is generally not based on extensive comparison testing. Ourguidelines for recommending a supplier are that (1) the particular brand has actually been found to be of superior quality, or (2) the item is difficult to find in the marketplace. An appendix lists the full names and addresses of all recommended suppliers, but these are by no means the only vendors of biological supplies. Readers may experiment with substituting their own favorite brands. Anyone carrying out these protocols will encounter the following hazardous or potentially hazardous materials: (1) radioactive substances, (2) toxic chemicals and carcinogenic or teratogenic reagents, (3) pathogenic and infectious biological agents, and (4) recombinant DNA. Most governments regulate the use of these materials; it is essential that they be used in strict accordance with local and national regulations. Cautionary notes are included in many instances throughout the manual, but we emphasize that users must proceed with the prudence and precaution associated with good laboratory practice, and that all materials be used in strict accordance with local and national regulations. Most of the protocols included in this manual are used routinely in our own laboratories. These protocols work for us; to make them work for you we have annotated critical steps and included critical parameters and troubleshooting guides in the commentaries to most units. However, the successful evolution of this manual depends upon readers' observations and suggestions. Consequently, a self-mailing, reader-response survey is included at the back of the manual (and is included with each supplement); we encourage readers to send in their comments. Electronic contact information is available at http://www.currentprotocols.com. Continuing to keep this manual up to date would be impossible without massive assistance from the Current Protocols staff at John Wiley & Sons. Among those who help us, we are extremely grateful to Joe Ingram, Virginia Chanda, Scott Holmes, Tom Cannon, Tom Downey, Susan Lieberman, Allen Ranz, and Joseph White. We are particularly indebted to Sarah Greene and her staff at Greene Publishing Associates who initially conceived of this project and helped to shape it with skill and unbounded patience. We are especially grateful to our co-workers who have helped with the manual by contributing material to it, by commenting on the chapters, or by field-testing the procedures. To those people—in our own labs, and in academic and industrial labs all over the world—we offer our deepest thanks.