Practical Methods in Molecular Biology PDF
Preview Practical Methods in Molecular Biology
Robert F. Schleif and Pieter C. Wensink Practical Methods in Molecular Biology With 49 Figures Springer-Verlag New York Heidelberg Berlin Robert F. Schleif Department of Biochemistry Brandeis University Waltham, Massachusetts 02254 U.s.A. Pieter C. Wensink Rosenstiel Center and Department of Biochemistry Brandeis University Waltham, Massachusetts 02254 U.s.A. Sponsoring Editor: Philip Manor Production: Kate Ormston Library of Congress Cataloging in Publication Data Schleif, Robert F. Practical methods in molecular biology. Bibliography: p. Includes index. 1. Molecular biology-Technique. I. Wensink, Pieter C. II. Title. [DNLM: 1. Molecular biology-Methods. QH 506 S339p] QH506.s34 574.8'8'028 81-9325 AACR2 © 1981 by Springer-Verlag New York Inc. Softcover reprint ofthe hardcover 1st edition 1981 All rights reserved. No part of this book may be translated or reproduced in any form without written permission from Springer-Verlag, 175 Fifth Ave nue, New York, New York 10010, U.s.A. The use of general descriptive names, trade names, trademarks, etc., in this publication, even if the former are not especially identified, is not to be taken as a sign that such names, as understood by the Trade Marks and Merchan dise Marks Act, may accordingly be used freely by anyone. 987654321 ISBN-13: 978-1-4612-5958-9 e-ISBN-13: 978-1-4612-5956-5 DOl: 10.1007/978-1-4612-5956-5 To our parents Preface This volume has evolved from a laboratory methods book that one of us first compiled nearly fifteen years ago. Since that time the book has undergone many minor revisions in order to include new methods and updated versions of older methods. The result has been an increasingly useful and more widely circulated book. However, the recent series of technological explosions generally lumped together under the name of "recombinant DNA technology" has been a turning point in the evolution of this previously underground publication. Minor revisions will no longer do. To keep the book useful we have had to make major revi sions and additions. The result is a dramatically expanded book that should be more useful to more people. The larger size and wider usefulness of the book have made this more formal publication seem a reasonable step to take. One of the reasons that this volume should be useful to many people is that it includes only procedures that have been used repeatedly by us and that have proven highly reliable both to ourselves and to others in our laboratories. We have made this decision to include only highly reliable recipes that we ourselves use because we want the book to be a dependable resource to others. While this dependabil ity is a major virtue of the book, it does force us to make a few sacrifices. We have had to exclude some important pro cedures. Also, as is frequently the case in experimental work, the most dependable procedures usually lack the sophisti cation or efficiency of refined, state-of-the-art methods. This book covers a broad range of techniques. The reci pes range from how to grow bacteria to techniques for clone selection by hybridization and for in vitro translation of messenger RNAs. Furthermore, we have presented them in a greater depth than is normally found in contemporary scientific publications. As a result, the procedures should be useful to someone with no prior experience with the techniques. viii Preface It is worth noting that the book contains a large number of recipes that are relevant to genetic engineering. Te ch niques are described for such routine manipulations as DNA purification, concentration, and quantitation as well as for some of the procedures necessary for cloning genes from higher organisms. However, this book does not include the very extensive collection of techniques for DNA sequencing which are described in Methods in Enzymology, Vol. 65, Part I, edited by Grossman and Moldave, 1980. It also does not contain the vast collection of detailed genetic techniques which are contained in Jeffrey Miller's book, Experiments in Molecular Genetics. The reader should use these sources for DNA sequencing or for extensive genetic analyses. The physical design of the book is intended to facilitate its use in the laboratory. The wide margins and hard but not glossy paper should allow users to pencil in and then to change their own modifications of the recipes. The clean and somewhat oversize type will ease reading by the harassed experimenter. This book could not have been written without the infor mation provided by a large number of people in the field. We are fortunate to be working in a field that has a tradition of free interchange of even the most intimate details of cur rent experimental methods. We thank all of those who have given us help in developing these recipes. Robert F. Schleif Pieter C. Wensink Contents Chapter 1 Using f. coli. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Strains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Strain Purity . . . . . . . .. ........... 1 Sources of Strains ............... 2 Commonly Used Strains . . . . . . . 2 Pedigrees and Genetic Maps . . . . . . . . . . . 2 Strains for Physiological Measurements. 2 Storage of Strains. . . . . . . . . . . . . . . . . . 3 Cell Growth . . . . . . . . . . . . . . . . . 3 Properties of Bacteria . 3 Phage Contamination 5 Use of Cells for Physiological Measurements . 6 Use of Cells for Genetic Purposes . . . . . . . . . . 7 Growing Cells for the Purification of Molecules. 9 Measuring Cell Density . . . . . . . . . . . . . . . . . . . . . . . . .. 9 Growing Large Quantities of Cells. 11 Opening Cells . . . . . . . . . .. .......... 15 Sonicating . 15 Grinding in Alumina . . . . . . . . . . . . . . . . . . . . . . . 15 Grinding with Glass Beads. . . . . . . . 15 Opening Cells with Lysozyme . . . . . . . . . . . . . . 16 Radiolabelling Cells. . . . . . . . . . . . 16 Nitrosoguanidine Mutagenesis. . ......... 17 Penicillin Selection . . . . . . . . . . . . . 18 Curing Cells of F-Factors . . . . . . . . . . . . . . . . 19 Curing with Acridine Orange ................ 19 Selecting Spontaneously Cured Cells. 20 Curing with Sodium Dodecyl Sulfate. . . . . . . . . . . . . 20 Curing with High Temperature. . . . . . . . . . . . . . . . . . .. 20 Making Cells Streptomycin-Resistant . . . . . . . . . . . . . . . .. 20 Crossing recA into Cells . .. ............... 21 Phage P1 Transduction of Genetic Markers ........... 22 Large-Scale Genetic Crosses. . . 24 Using Transposons in Strain Construction. 26 x Contents Chapter 2 Bacteriophage Lambda ................... 27 Two Useful Mutants. . . . . . . . . . . . . . . . . . 27 Cl857 . . . . . . . . . . . . . . . . . . . . . . . . ... 27 S7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Titering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Growing Plate Stocks ... . . . . . . . . . . . . . . . . . . . . . . . . .. 29 Large-Scale Growth in Liquid . . . . . . . . . . . . . . . . .. 31 Purification . . . . . . . . . . . . . . . . . . . . . . 33 Genetic Crosses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 35 Scoring Plaques . . . . . . . . . . . . . . . . . . . . . . . .. 36 Making Strains Lambda-Resistant . . . . . . . . . . . . . . . . . . 37 Testing Colonies for the Ability to Grow Lambda . . . . .. 38 Making Ly sogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Testing Lysogen Candidates . . . . . . . . . . . . . . . . . . .. 40 Streaking for Single Plaques . . . . . . . . . . . . . . . 40 Selecting Deletions .. . . . . . . . . . . . 41 Chapter 3 Enzyme Assays ......................... 43 ,B-Galactosidase . . . . . . . . . . . . . . . . . . . . . . . 43 RNA Polymerase .... .......... . . . . . . . . . . .. 45 Arabinose Isomerase. . . . . . . . . . . . . . . . . . 46 Lysozyme . . . .. .......... . . . . . .. 50 Ribulokinase . . . . . . . . . 52 E. coli-Coupled Transcription-Translation System. 56 Chapter 4 Working with Proteins .................. . 61 Ammonium Sulfate Precipitation of Proteins. . . . . . . . 62 Removing Nucleic Acids by Phase Partition ........ 64 Bulk Separation of Proteins from Nucleic Acids . 65 Purification of Protein-Bound DNA or RNA. 67 Columns, Fraction Collectors, and Plumbing 68 Ion Exchange Chromatography and Gel Filtration 69 Preparing Ion Exchange Resins . 69 Pouring Columns . . . . . . . . . . . . . . 70 Loading and Eluting Columns . . . . . . . . . . .. 71 Determining Protein Concentration. . . 74 Optical Density . . . . . . . 74 Biuret. . . . . . . . . . . . . 74 Lowry. . ................................. 75 Lowry for Dilute Samples . . . . . . . . . . . . . . . . . . . .. 75 Concentrating Protein Solutions. . . . . . . . . . . . . . . . . . . .. 76 Stabilizing Proteins. . . . . . . . . . . . . . . . . . . . . . .. 77 Polyacrylamide Gel Electrophoresis of Proteins . . . . . . .. 78 Making a Gel Sandwich. . . . . . . . . . . . . . . . . . . . . . . . .. 80 SDS-10% Acrylamide Gel with Stacking Gel. . 81 Urea-SDS-Acrylamide Gradient Gels. . 84 Staining Gels. . . . . . . . . . . . . . . . . . . . . . . . . 87 Fluorography of [3H]- or [35S]-Labelled Proteins in Acrylamide Gels. . . . . . . . . . . . . . . . . . . . . . . . . 87 Recovering PPO from Solution in DMSO 88 Contents xi Chapter 5 Working with Nucleic Acids ............. . 89 Measuring Nucleic Acid Concentration and Purity. 89 Optical Methods .... 89 Fluorescence Method ........ . 90 Storing DNA .... . ...... 92 Cleaning DNA 93 Cleaning by DEAE ...... . 94 Cleaning by Hydroxyapatite. 94 Precipitating DNA with Ethanol 95 TCA Precipitation Assay .. 96 Precipitation and Size Fractionation of DNA with Poly- ethylene Glycol ...... . 97 Isolating E. coli DNA . 98 Isolating Lambda DNA. 99 Phenol Extraction. 99 SDS Extraction . 100 Isolating Plasmid DNA .................. . 101 Large-Scale Plasmid Isolation 106 Isolating Drosophila DNA .................. . 108 Preparing Nucleoside Triphosphate Solutions. 111 Chromatographic Analysis of Nucleosides . 112 Gel Electrophoresis of DNA ................ . 114 Agarose Gel Electrophoresis. 115 Polyacrylamide Gel Electrophoresis . 120 Staining and Photographing Gels. 121 Extracting DNA from Acrylamide and Agarose Gels 122 Mapping Restriction Endonuclease Sites on DNA. 125 Chapter 6 Constructing and Analyzing Recombinant DNA .............................. . 129 Joining the Ends of DNA Molecules. 129 Making Blunt Ends by S1 Digestion 130 Ligation with T 4 DNA Ligase. ........ 130 Lambda Exonuclease Digestion to Yield Free 3' Ends. 132 Addition of Homopolymer Tails. 133 E. coli Transformation with Plasmid DNA 134 Storing Strains that Contain Plasm ids . 136 Cycloserine Selection of Recombinant Plasm ids . 136 In Vitro Radiolabelling of DNA and RNA. . 137 Radiolabelling DNA by Nick Translation 138 Synthesizing Radiolabelled RNA Complementary to DNA .......... . 140 Synthesizing Radiolabelled DNA Complementary to RNA ............... . 141 Radiolabelling the 5' Ends of RNA or DNA ... 143 General Aspects of Nucleic Acid Hybridization Reactions . . . . . . . . . . ..... . 145 Screening Recombinant DNA Clones by Nucleic Acid Hybridization . . . ..... . 146 Screening Phage Plaques. 147 Screening Colonies 149 xii Contents Isolating DNA from a Single Colony .. . ........ 151 Southern Transfers .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 152 Selecting RNA Complementary to a DNA. . . . . . . .. 156 Preparing DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 157 Preparing NBM Paper . . . . . . . . . . . . . . . . . . . . . .. 157 Converting NBM Paper to DBM Paper and Binding the DNA ................................... 158 Hybridizing RNA to DBM-Bound DNA. . .. 158 Recovering the RNA . . . . . . . . . . . . . . . . .. 159 Synthesis of NBPC (nitrobenzylpyridinium chloride) .. 159 In Vitro Translation Systems from Higher Organisms ... 161 Preparing a Wheat Germ Extract . . . . . . . . . . . . . . . .. 161 Wheat Germ Translation Reaction. . . . . . . . . . . . . . .. 163 Preparing a Rabbit Reticulocyte Lysate. . . . . . . . . . .. 163 Micrococcal Nuclease Digestion of Lysate. . . . . . .. 164 Reticulocyte Translation Reaction .. . . . . . . . . . . . .. 165 Purifying Total and Polysomal RNA. . . . . . . . . . . . . 166 Extracting RNA from Drosophila Adults . . . . . .. 166 Extracting Polysomal RNA from Drosophila Embryos. 167 Purification of Poly-A + (mRNA-Enriched) RNA . . . 168 RNA Size Fractionation by Sucrose Gradient Centrifugation ........ . . . . . . . . . .. 170 Making the Sucrose Gradients. . 171 Preparing the RNA. . . . . . . . . . . .. . .............. 171 Sedimenting and Collecting the RNA. . . . . . . . . . . 172 Chapter 7 Assorted Laboratory Techniques ......... 173 Glass and Plastic Containers. . . . . . . . . . . . . . . . 173 Siliconizing Glassware. . . . . . . . . . . . . . . . . 174 Washing Pipettes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 174 pH Meters . . . . . . . . . . . . . . . . . . . . . . . 176 Buffers (Tris, Phosphate, Good Buffers, Cacodylate) . .. 177 Beckman Ultracentrifuges. . . . . 178 Filling and Balancing Tubes ........... 178 Loading Rotors. . . . . 179 Drawing Figures. . . . . . . . . . . . . . . . 179 Slides and Negatives . . . . . . . . . . . . . .. ............ 181 Polaroid Slides ................................ 181 Conventional Slides and Negatives. . . . . . . . . . . . 181 Film Sensitometry ................... . . . . . . . .. 183 Autoradiography and Fluorography ................ 184 Dialysis Tubing. . . . . . . . . . . . . . . . . . . . . . . . . . . . 186 Distilling Phenol. . . . . . . . . . . . . . ............... 187 Recovering Used CsCl ......................... 188 Sources of Chemicals. . . .................... 189 Hazards and Cautions ............................ 190 Desiccators, Preparative Centrifuges, and Vacuum ~m~ ................................... . 190 Chemicals ............................... . 191 Electrical Hazards and Protections ............... . 192
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