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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
x Contents
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
xii
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
xx
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