Essential Molecular Biology

Volume One

Second Editio n

T. A. Brown

List of protocols page xv

Abbreviations xix

1 Getting started in molecular biology 1T. A. Brown

1 Introduction 1

2 Practical requirements for molecular biology research 2Experimental skills 2Equipment 2A word on kits 5

3 Health hazards and safety procedures 5Microbiological safety 5Radiochemicals 6Chemical hazards 6Ultraviolet radiation 7High-voltage electricity 7

4 Research strategies for molecular biology 8Gene cloning in outline 8PCR in outline 1 0The choice between cloning and PCR 1 0Basic techniques needed for cloning and PCR 1 2

5 Planning an informative project 1 6Will it be possible to identify the correct clone? 1 6Will the DNA sequence provide any new information? 17Will studies of gene expression be informative? 1 7Conclusion 1 8

References 18

2 Microbiological techniques for molecular biology : bacteria andphages 2 1Brian W. Bainbridge

1 Introduction: techniques for handling microbes 21Basic microbiological techniques 21

Basic techniques of microbial genetics 22

Safety in the molecular biology laboratory 22

Sterilization and disinfection 23

Basic principles of aseptic technique 24

2 Culturing of Escherichia coli 2 5

Single-colony isolation 2 7

Small-scale broth culture 29

Large-scale broth culture 30

3 Characterization of bacterial strains 3 2

Genotypes and strain nomenclature 3 2

Characterization of nutritional mutants 3 4

Characterization of antibiotic resistance 3 5

Characterization of recA and UV-sensitive mutants 3 7

Characterization of the utilization of lactose : X-gal 3 7

Detection of lysogeny 38

Screening for plasmids 38

Microscopic examination of cultures 3 9

4 Preservation of stock cultures 40

Preservation of short-term cultures 40

Stab cultures 4 1

Preservation of cultures with glycerol or DMSO 4 1

Freeze-dried cultures 42

5 Culturing of bacteriophages k and M13 42

Theoretical background 42

Factors affecting the growth and survival of phage k 43

General techniques for the assay of phages by the plaque method 44

Purification of phage by single-plaque isolation 4 6

Preparation of small-scale phage stocks 46

Preparation of large-scale phage stocks 4 8

Purification of k phage particles 4 8

Induction of X lysogens 5 0

Techniques involving phage M13 5 1

Methods for preserving phage stocks 5 1

6 Troubleshooting 5 1

General principles 5 1

Contamination 52

Poor growth of bacteria or phage 5 2

References 5 3

3 Purification of DNA 55

Paul Towner

1 Introduction 55

2 Silica-based methods for the isolation of DNA 55

Description of the methodology 55

Purification of genomic and viral DNA 5 7

Plasmid DNA 6 0

Post-purification treatment of samples with phenol 6 4

3 Assessment of quality 66

References 67

4 Purification of RNA 69

Miles Wilkinso n

1 Introduction 6 9

2 Ribonuclease-free conditions 69

3 Quantification of RNA 7 1

4 Precipitation and storage of RNA 7 1

5 Preparation of total cellular RNA 72

6 Cytoplasmic and nuclear RNA 79

Cytoplasmic RNA 79

Nuclear RNA 83

7 Poly(A) + RNA 8 4

Acknowledgements 8 7

References 8 7

5 Nucleic acid electrophoresis in agarose gels 89

Douglas H. Robinson and Gayle J. Laflech e

1 Introduction 8 9

Voltage, current and power : interactive effects on gel electrophoresis 8 9

Physical chemistry of agarose 9 1

2 Preparation of agarose gels 9 4

Buffer systems 94

Dissolving agarose 95

Casting an agarose gel 9 7

Loading and running DNA in an agarose gel 9 8

Detection of DNA in agarose gels 10 0

Alkaline gel electrophoresis 10 5

Drying an agarose gel 10 6

3 Vertical agarose gels and fast-running horizontal gels 10 8

Vertical gels 108

Fast-running horizontal gels 11 1

4 Electrophoresis of RNA 11 2

Denaturing systems 11 2

Preparation of RNA samples 11 3

Electrophoresis of RNA 11 5

References 11 9

6 Recovery of DNA from electrophoresis gels 12 1

Paul Towner

1 Introduction 12 1

2 Agarose gels 122

Recovery of DNA from agarose gels 122

Limitations on DNA recovery from agarose 12 6

3 Polyacrylamide gels 12 6

Recovery of DNA from polyacrylamide gels 12 7

Acknowledgement 12 8

References 128

7 Construction of recombinant DNA molecules 129

Richard Powell and Frank Ganno n

1 Introduction 12 9

2 Restriction enzyme digestions 13 0

3 Preparation of vectors for molecular cloning experiments 13 7Phage X 13 8Cosmids 14 0Plasmids 14 1

4 Construction of recombinant molecules 142Ligation 142Linkers and adapters 14 5Tailing 14 7TEA cloning of PCR products 149

5 Conclusion 149

Acknowledgements 149

References 15 0

8 Generation and identification of recombinant clones 15 1

T. A. Brown

1 Introduction 15 1

2 Introduction of DNA into E. coli cells 15 1Uptake of DNA by chemically treated cells 152DNA uptake by electroporation 15 7In vitro packaging 158

3 Plating out and recombinant selection 161Selection of plasmid vectors carrying antibiotic resistance genes 16 2Lac selection of plasmids 164Recombinant selection with M13 vectors 166Recombinant selection with X vectors 168

4 Vectors combining features of both plasmids and phages 169Cosmids 17 1Phagemids 17 1

References 173

9 Survey of cloning vectors for Lscherichia coil 175

T. A. Brown

1 Introduction 175

2 E. coll. as the host organism for recombinant DNA research 176Advantages of E. coli as a host organism 17 6E. coli is not an ideal host for gene cloning 177

3 Plasmid vectors 178General properties of plasmid vectors 179Basic plasmid vectors 18 1Phagemids 184RNA expression vectors 18 6Protein expression vectors 188

Cosmids 19 1

Cloning vectors for PCR products 19 5

Multipurpose vectors 197

4 Bacteriophage A vectors 197

Practical considerations relevant to the use of K vectors 198

Examples of X. vectors 201

5 Bacteriophage M13 vectors 204

General properties of M13 vectors 20 4

Acknowledgements 206

References 20 6

Appendices

Al Important Escherichia coil strains 209

A2 Recipes and general procedures 215

A3 Restriction endonucleases 22 1

A4 DNA and RNA modification enzymes 229

A5 List of suppliers 23 3

Index 237