Experiments inMolecular Biology

Biological Methods

Experiments in Molecular Biology, edited byRobert J. Slater, 1986

Microcharacterization 0/ Polypeptides, editedby John E. Shively, 1986

Methods in Molecular Biology, edited by JohnM. Walker, 1984

Volume I: ProteinsVolume II: Nucleic Acids

Liquid Chromatography in Clinical Analysis,edited by Pokar M. Kabra and Laurence J.Marton, 1981

Metal Carcinogenesis Testing, by Max Costa,1980

Experiments•In

Molecular Biology

Edited by

Robert J. Slater

Humana Press • Clifton, New Jersey

Library of Congress Cataloging in Publication DataMain entry under title:

Experiments in molecular biology

(Biological methods)Includes index.

1. Molecular Biology-Experiments. I. Slater,Robert J. II. SeriesQH506.E96 1985 574.87'382 85- 21882ISBN 0- 89603- 082- 2© 1986 The Humana Press Inc.Crescent ManorPO Box 2148Clifton, NJ 07015

All rights reserved

No part of this book may be reproduced, stored in a retrievalsystem, or transmitted in any form or by any means, electronic,mechanical, photocopying, microfilming, recording, or otherwisewithout written permission from the Publisher.

Printed in the United States of America

PREFACE

Research in the field of molecular biology has progressed ata fascinating rate in recent years. Much of this progressresults from the development of new laboratory techniquesthat allow very precise fractionation and analysis of nucleicacids and proteins, as well as the construction of recom­binant DNA molecules that can then be cloned andexpressed in host cells. Progress has been so rapid that therehas been a shortfall in the training of appropriately qualifiedstaff. Many existing laboratory workers require retraining,and many educational institutions have had difficulty incor­porating the new molecular biology techniques into theirteaching programs. Although there are several manualscurrently available that describe laboratory techniques inmolecular biology, they are principally written for the indivi­dual research worker and are not intended for use in thedesign of practical classes for students.

The aim of this book is to provide just such a series of pro­tocols for the teaching of practical molecular biology. Theidea arose following the success of several Workshops inMolecular Biology, organized and taught by staff in the Biol­ogy Department of the Hatfield Polytechnic. Gradually, theprotocols used in the workshops have been incorporated intothe Hatfield undergraduate and postgraduate teaching pro­grams and have now been collected together to form a book.The lecturers and demonstrators in these workshops andcourses, together with respected authorities from other insti­tutions, have contributed their tried and tested protocols tothis book so that others may more readily incorporatematerial central to modem molecular biology into their own

v

vi Preface

undergraduate and beginning graduate-level courses, orindeed even develop similar laboratory workshops in theirown institutions.

The chapters following describe a range of the mostimportant techniques in molecular biology research today.The book begins with chapters on the extraction and mani­pulation of DNA, continues with articles on RNA and pro­tein techniques, and closes with some experiments on wholecells. In all but the first chapter, which is intended as anintroductory experiment, the procedures described are thosenormally used in the research laboratory and have not beensimplified. This was done because the objectives here are toteach participants in the experiments how the techniques areroutinely used and to provide a source book of proceduresthat will be directly useful in their subsequent careers.Nevertheless, the level of detail given is such that everyexperiment should prove successful at the first attempt.

Each chapter describes an experiment based on a particu­lar technique in molecular biology, and all are presented inthe same format. The Introduction gives a brief summary ofthe techniques to be employed and describes the importanceof such techniques to current research. The aims and objec­tives of the experiment are then described and an indicationis given of the time required to complete the practical pro­cedures. The Materials Provided section lists all those itemsthat should be available before the practical work begins.This is not intended to give all the details concerning theequipment or buffers and solutions required, but is a check­list intended to help organization of the experiment andensure that nothing has been forgotten. The Protocol sectiondescribes the practical procedure in detail, in numberedchronological steps. When an experiment involves a numberof separate stages, perhaps involving more than one session,the Protocol is divided into discrete sections as appropriate.The Results and Discussion section describes the predictedresults and gives details of any data manipulations that arerequired. In many cases, interesting discussion points areraised, or questions about data interpretation are posed.

This book contains two Appendices. The first of these,Appendix I, supplies general information relevant to manyof the preceeding chapters. Appendix II, on the other hand,is divided into sections related to individual chapters and is

Preface vii

concerned with the preparatory work required to set upeach experiment. Appendix II is self-contained anddescribes in detail any procedures that must be completedprior to the practical class, lists the special equipmentrequired, and the amounts of materials, solutions, andbuffers needed for classes of 20 participants, who will beassumed to work in pairs unless otherwise indicated. Someexperiments are best suited to smaller groups, whereas oth­ers can be expanded to teach much larger classes; this isindicated where appropriate. It is assumed that basiceveryday laboratory equipment, such as pipets, bench­centrifuges, ice buckets, and waterbaths or incubators willbe available; these are often not listed.

Each chapter in the book has been written in a style suit­able for both teacher and student. If the reader is a teacherof molecular biology, he or she should first select theexperiments of interest; the book can then be followed sys­tematically to form a whole course on the techniques ofmolecular biology, or individual chapters can be chosen asrequired. The Introduction will give an indication of thetime required to carry out the procedures, and Appendix IIcan be given to the laboratory technician to prepare for thepractical class. The participants should first read the Intro­duction to become familiar with the experiment's purpose.Then, before commencing the practical work, they shouldread the Materials Provided section to be sure that all thenecessary items have been supplied. The experiment maythen proceed by following the steps described in the Proto­col. Finally, when the course has been completed, we hopeand expect that the participants will always feel able torefer to the Protocol section and Appendix II as a resourcefor future work in the field.

Robert J. Slater

Contents

Preface v

CHAPTER 1DNA Isolation and Transformation in

Escherichia coli: An Introduction

Elliot B. Gingold

1. Introduction............................................................... 1

2. Materials Provided................................................... 32.1. Cultures 32.2. Solutions.......................................................... 32.3. Growth Media................................................. 32.4. Equipment 3

3. Protocol...................................................................... 33.1. DNA Isolation................................................. 43.2. Preparing the E. coli Recipient....................... 53.3. Transformation................................................ 5

4. Results and Discussion............................................ 6

CHAPTER 2

Isolation of Plasmid DNA: The Cleared Lysate andesCI Gradient Technique

Stephen A. Boffey

1. Introduction............................................................... 7

2. Materials Provided................................................... 102.1. Solutions and Chemicals 102.2. Equipment 10

ix

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3. Protocol.............. 10

4. Results and Discussion 14

Reference.......................................................................... 15

CHAPTER 3Restriction Endonuclease Digestion and

Agarose Gel Electrophoresis of DNAStephen A. Boffey

1. Introduction............................................................... 17

2. Materials Provided................................................... 202.1. Solutions and Chemicals 202.2. Equipment 20

3. Protocol..... 203.1. Restriction of DNA 203.2. Casting of Agarose Gel..... 213.3. Loading and Running Gel............................. 23

4. Results and Discussion 24

References........................................................................ 27

CHAPTER 4Restriction Site MappingAlan Hall

1. Introduction 29

2. Materials Provided................................................... 322.1. Solutions 322.2. Agarose Gel Electrophoresis......................... 332.3. Apparatus and Equipment 33

3. Protocol......... 333.1. Day 1................................................................ 333.2. Day 2. 35

4. Results and Discussion 36

Reference.......................................................................... 38

Contents

CHAPTERS

Southern BlottingAlan Hall

xi

1. Introduction............................................................... 391.1. Transfer Step 411.2. Preparation of Radioactive DNA.................. 411.3. Hybridization Step. 421.4. Washing Step.................................................. 43

2. Materials Provided................................................... 442.1. Electrophoresis................................................ 442.2. Southern Blotting...................................... 442.3. Hybridization and Washing.......................... 442.4. Nick Translation. 44

3. Protocol................................................................. 453.1. Day 1................................................................ 453.2. Day 2.................................................... 483.3. Day 3.. 493.4. Nick Translation Procedure..... 49

4. Results and Discussion 52References 52

CHAPTER 6

Preparative Electrophoresis: Extraction of NucleicAcids From Gels

Alan Hall

1. Introduction............................................................... 551.1. Extraction From Acrylamide Gels 551.2. Extraction From Agarose Gels.......... 56

2. Materials Provided... 582.1. Solutions for Digestion.................................. 582.2. Electrophoresis................................................ 582.3. Gel Extraction 58

3. Protocol........ 583.1. Day 1................................................................ 583.2. Day 2................................................................ 61

4. Results and Discussion.......... 62

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CHAPTER 7DNA Ligation and Escherichia coli

TransformationAlan Hall

Contents

1. Introduction............................................................... 63

2. Materials Provided. 672.1. Solutions for Digestion.................................. 672.2. Solutions for Ligation..................................... 672.3. Preparation of Competent Cells and

Transformation................................................ 68

3. Protocol..... 68

4. Results and Discussion........ 71

CHAPTER 8Detection of Plasmid By Colony HybridizationElliot B. Gingold

1. Introduction............................................................... 73

2. Materials Provided 762.1. Cultures 762.2. Solutions 762.3. Equipment 77

3. Protocol. 773.1. Chloramphenicol Amplification 773.2. Lysis and Filter Binding................................. 783.3. Hybridization 783.4. Washing........ 793.5. Autoradiography............................................. 79

4. Results and Discussion 80

Reference.......................................................................... 80

Contents xiii

CHAPTER 9

Microisolation and Microanalysis of Plasmid DNAElliot B. Gingold

1. Introduction............................................................... 81

2. Materials Provided 822.1. Cultures 822.2. Solutions 832.3. Equipment 83

3. Protocol...................................................................... 833.1. DNA Isolation................................................. 833.2. Minigel Analysis............................................. 84

4. Results and Discussion 85

CHAPTER 10Isopycnic Centrifugation of DNA: Extraction and

Fractionation of Melon Satellite DNA byBuoyant Density Centrifugation in CesiumChloride Gradients

Donald Grierson

1. Introduction............................................................... 87

2. Materials Provided................................................... 912.1. Plant Material.................................................. 912.2. Solutions and Chemicals 912.3. Apparatus and Equipment............................ 91

3. Protocol...................................................................... 91

4. Results and Discussion............................................ 97

References 98

xiv Contents

CHAPTER 11Purification of Plant Ribosomal DNA: Use of

Buoyant Density Centrifugation in CesiumChloride-Actinomycin D Gradients andDNA-RNA Hybridization

Donald Grierson

1. Introduction............................................................... 99

2. Materials Provided................................................... 1002.1. DNA Isolation and Buoyant Density

Centrifugation 1002.2. DNA-RNA Hybridization 100

3. Protocol....... 101

4. Results and Discussion 105

References .. 106

CHAPTER 12The Extraction and Affinity Chromatography of

Messenger RNARobert J. Slater

1. Introduction............................................................... 109

2. Materials Provided 1122.1. Day 1................................................................ 1122.2. Day 2................................................................ 112

3. Protocol...................................................................... 1133.1. Day 1: The Extraction of Total Nucleic

Acids................................................. 1133.2. Day 2: To Determine the Concentration

and Specific Radioactivity of theNucleic Acid Preparation 115

3.3. Day 2: Oligo(dT)-CelluloseChromatography............................. 116

4. Results and Discussion 117

5. Notes 118

References 119

Contents

CHAPTER 13

Agarose Gel Electrophoresis of RNARobert J. Slater

xv

1. Introduction............................................................... 121

2. Materials Provided................................................... 1232.1. Solutions and Chemicals 1232.2. Equipment 123

3. Protocol............ 1243.1. Casting of Agarose Gel.................................. 1243.2. Loading and Running Gel............................. 125

4. Denaturing Gels........................................................ 1264.1. Introduction..................................................... 1264.2. Materials Provided......................................... 1274.3. Protocol............................................................ 127

5. Results and Discussion 127

References........................................................................ 129

CHAPTER 14In Vitro Protein Synthesis

Jayne A. Matthews and Raymond A. McKee

1. Introduction............................................................... 131

2. Materials Provided................................................... 1342.1. Expt. I, Preparing the Wheat Germ

Lysate 1342.2. Expt. 2, Optimizing the Magnesium

Concentration for the WheatGerm Lysate................................... 135

2.3. Expt. 3, Translation for Analysis ofProducts.......................................... 136

3. Protocol.............. 1373.1. Expt. I, Day 1. Preparing the Wheat

Germ Lysate.................... 1373.2. Expt. 2, Day 1. Optimizing the Magnesium

Concentration for theWheat Germ Lysate 138

xvi Contents

3.3. Expt. 3, Day 2. Translation for Analysisof Products 141

4. Results and Discussions 143

References 146

CHAPTER 15

SDS Polyacrylamide Gel Electrophoresis ofProteins: Determination 0/ Protein MolecularWeights and High-Resolution Silver Staining

John M. Walker

1. Introduction............................................................... 149

2. Materials Provided................................................... 1532.1. SDS Gel Electrophoresis................................ 1532.2. Silver Staining 153

3. Protocol......... 1543.1. 50S Gel Electrophoresis................................ 1543.2. Silver Staining................................................. 158

4. Results and Discussion... 158

References 159

Further Reading............................................................... 160

CHAPTER 16Isoelectric Focusing (IEF) of Proteins in Thin­

Layer Polyacrylamide GelsJohn M. Walker

1. Introduction............................................................... 161

2. Materials Provided................................................... 163

3. Protocol........... 163

4. Results and Discussion. 166

References 169

Further Reading............................................................... 169

Contents

CHAPTER 17Protein (Western) Blotting With

ImmunodetectionJohn M. Walker

xvii

1. Introduction............................................................... 171

2. Materials Provided................................................... 175

3. Protocol...................................................................... 175

4. Results and Discussion............................................ 177

References........................................................................ 177

Further Reading 178

CHAPTER 18Thin-Layer Gel Filtration: Determination of

Protein Molecular Weights

John M. Walker

1. Introduction............................................................... 179

2. Materials Provided 181

3. Protocol...................................................................... 181

4. Results and Discussion............................................ 183

References 184

CHAPTER 19

The Dansyl-Edman Method for PeptideSequencing

John M. Walker

1. Introduction............................................................... 1851.1. The Coupling Reaction 1851.2. The Cleavage Reaction................................... 186

2. Materials Provided................................................... 1892.1. The Edman Degradation................................ 1892.2. The Dansyl Method....................................... 189

xviii Contents

3. Protocol............................ 189

4. Results and Discussion 194

References 197

Further Reading 197

CHAPTER 20

The Double Immunodiffusion Technique:Immunoprecipitation and Analysis of AntigenicProtein in Gel

David L. Cohen

1. Introduction............................................................... 199

2. Materials Provided................................................... 2032.1. Day 1................................................................ 2032.2. Day 2. 204

3. Protocol... 2043.1. Day 1................................................................ 2043.2. Day 2................................................................ 207

4. Results and Discussion 2084.1. General Interpretation of Precipitation

Patterns............................................................ 2094.2. Likely Results of Gel Diffusion Expts. 1-4. 210

References 211

CHAPTER 21Immunoelectrophoretic Techniques:

2-D Immunoelectrophoresis andRocket Immunoelectrophoresis

John M. Walker

1. Introduction............................................................... 213

2. Materials Provided 2162.1. Rocket Immunoelectrophoresis 2162.2. 2-D Immunoelectrophoresis.......................... 2162.3. General............................................................. 216

Contents xix

3. Protocol...................................................................... 2173.1. Rocket Immunoelectrophoresis 2173.2. 2-D Immunoelectrophoresis.......................... 221

4. Results and Discussion............. 2234.1. Rocket Immunoelectrophoresis 2234.2. 2-D Immunoelectrophoresis.......................... 223

References 224

Further Reading............................................................... 225

CHAPTER 22

The Time Course of fJ-GalactosidaseInduction in Escherichia coli

Kenneth H. Goulding

1. Introduction............................................................... 227

2. Materials Provided................................................... 2302.1. Equipment 2302.2. Reagents........................................................... 230

3. Protocol...................................................................... 2314. Results and Discussion 234References 235

CHAPTER 23

Experiments on Enzyme Induction in theUnicellular Green Alga Cblorella fusca

Kenneth H. Goulding

1. Introduction............................................................... 237

2. Materials Provided................................................... 2402.1. Equipment....................................................... 2402.2. Reagents........................................................... 2412.3. Enzyme Preparations...................................... 241

3. Protocol...................................................................... 241

4. Results and Discussion............... 243

References 246

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CHAPTER 24

Yeast Protoplast FusionElliot B. Gingold

Contents

1. Introduction............................................................... 247

2. Materials Provided 2492.1. Strains 2492.2. Solutions .. 2492.3. Media .. 2492.4. Equipment .. 249

3. Protocol.... 2503.1. Protoplast Formation...................................... 2503.2. Protoplast Fusion. 2513.3. Protoplast Regeneration................................. 252

4. Results and Discussion 252

References 253

CHAPTER 25

The Ames TestElliot B. Gingold

1. Introduction............................................................... 255

2. Materials Provided................................................... 2602.1. Cultures 2602.2. Media............................................................... 2602.3. Additives.......................................................... 2602.4. Mutagens 2602.5. Equipment 260

3. Protocol..... 260

4. Results and Discussion.. 262

References 263

ApPENDIX I

Glassware and Plasticware 265Preparation of Buffers and Solutions 266Quantitation of Nucleic Acids 267

Contents xxi

Quantitation of Proteins 268Safe Handling of Microorganisms............................ 270Safe Handling of Radioactivity 272Autoradiography.......................................................... 274Using an Ultracentrifuge 280The Plasmid pBR322 281

ApPENDIX II

Details and Notes on Preparation........... 283

Index................................................................................ 371

Contributors

STEPHEN A. BOFFEY • Division of Biological and Environ-mental Studies, The Hatfield Polytechnic, Hertfordshire, UK

DAVID L. COHEN • Division of Biological and Environmen-tal Studies, The Hatfield Polytechnic, Hertfordshire, UK

ELLIOT B. GINGOLD • Division of Biological and Environmen­talStudies, The Hatfield Polytechnic, Hertfordshire, UK

KENNETH H. GOULDING • Division of Biological and Environ­mental Studies, The Hatfield Polytechnic, Hertfordshire, UK

DONALD GRIERSON • Department of Physiology and Environ­mental Sciences, University of Nottingham, Loughborough,UK

ALAN HALL • Institute of Cancer Research, Chester BeattyLaboratories, London, UK

RAYMOND A. McKEE • Department of Botany, University ofLeicester, Leicester, UK

JAYNE A. MATTHEWS • Department of Clinical Chemistry,Wolfson Research Laboratories, Queen Elizabeth MedicalCentre, Birmingham, UK

ROBERT J. SLATER • Division of Biological and Environmen-tal Studies, Th» Hatfield Polytechnic, Hertfordshire, UK

JOHN M. WALKER • Division of Biological and Environmen-tal Studies, The Hatfield Polytechnic, Hertfordshire, UK

xxii