9780199636426

Essential Molecular Biology A Practical Approach Volume I

by
  • ISBN13:

    9780199636426

  • ISBN10:

    0199636427

  • Edition: 2nd
  • Format: Paperback
  • Copyright: 2000-12-28
  • Publisher: Oxford University Press

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Summary

The two Essential Molecular Biology books in the Practical Approach Series are designed for the absolute beginner at gene cloning whether they be at the start of their career or an experienced researcher in another field. As with the first editions, the objective of both volumes is to combine solid practical information with sufficient background material to ensure that the novice can understand how a technique works, what it achieves, and how to make modifications to suit personal requirements. Volume 1 concentrates on the procedures for DNA and RNA manipulation: purification, electrophoresis, and the construction and cloning of recombinant molecules. It also includes a general introduction to molecular biology in the laboratory and a survey of cloning vectors for Escherichia Coli .

Table of Contents

List of protocols
xv
Abbreviations xix
Getting started in molecular biology
1(20)
T. A. Brown
Introduction
1(1)
Practical requirements for molecular biology research
2(3)
Experimental skills
2(1)
Equipment
2(3)
A word on kits
5(1)
Health hazards and safety procedures
5(3)
Microbiological safety
5(1)
Radiochemicals
6(1)
Chemical hazards
6(1)
Ultraviolet radiation
7(1)
High-voltage electricity
7(1)
Research strategies for molecular biology
8(8)
Gene cloning in outline
8(2)
PCR in outline
10(1)
The choice between cloning and PCR
10(2)
Basic techniques needed for cloning and PCR
12(4)
Planning an informative project
16(5)
Will it be possible to identify the correct clone?
16(1)
Will the DNA sequence provide any new information?
17(1)
Will studies of gene expression be informative?
17(1)
Conclusion
18(1)
References
18(3)
Microbiological techniques for molecular biology: bacteria and phages
21(34)
Brian W. Bainbridge
Introduction: techniques for handling microbes
21(4)
Basic microbiological techniques
21(1)
Basic techniques of microbial genetics
22(1)
Safety in the molecular biology laboratory
22(1)
Sterilization and disinfection
23(1)
Basic principles of aseptic technique
24(1)
Culturing of Escherichia coli
25(7)
Single-colony isolation
27(2)
Small-scale broth culture
29(1)
Large-scale broth culture
30(2)
Characterization of bacterial strains
32(8)
Genotypes and strain nomenclature
32(2)
Characterization of nutritional mutants
34(1)
Characterization of antibiotic resistance
35(2)
Characterization of recA and UV-sensitive mutants
37(1)
Characterization of the utilization of lactose: X-gal
37(1)
Detection of lysogeny
38(1)
Screening for plasmids
38(1)
Microscopic examination of cultures
39(1)
Preservation of stock cultures
40(2)
Preservation of short-term cultures
40(1)
Stab cultures
41(1)
Preservation of cultures with glycerol or DMSO
41(1)
Freeze-dried cultures
42(1)
Culturing of bacteriophages λ and M13
42(9)
Theoretical background
42(1)
Factors affecting the growth and survival of phage λ
43(1)
General techniques for the assay of phages by the plaque method
44(2)
Purification of phage by single-plaque isolation
46(1)
Preparation of small-scale phage stocks
46(2)
Preparation of large-scale phage stocks
48(1)
Purification of λ phage particles
48(2)
Induction of λ lysogens
50(1)
Techniques involving phage M13
51(1)
Methods for preserving phage stocks
51(1)
Troubleshooting
51(4)
General principles
51(1)
Contamination
52(1)
Poor growth of bacteria or phage
52(1)
References
53(2)
Purification of DNA
55(14)
Paul Towner
Introduction
55(1)
Silica-based methods for the isolation of DNA
55(11)
Description of the methodology
55(2)
Purification of genomic and viral DNA
57(3)
Plasmid DNA
60(4)
Post-purification treatment of samples with phenol
64(2)
Assessment of quality
66(3)
References
67(2)
Purification of RNA
69(20)
Miles Wilkinson
Introduction
69(1)
Ribonuclease-free conditions
69(2)
Quantification of RNA
71(1)
Precipitation and storage of RNA
71(1)
Preparation of total cellular RNA
72(7)
Cytoplasmic and nuclear RNA
79(5)
Cytoplasmic RNA
79(4)
Nuclear RNA
83(1)
Poly(A)+ RNA
84(5)
Acknowledgements
87(1)
References
87(2)
Nucleic acid electrophoresis in agarose gels
89(32)
Douglas H. Robinson
Gayle J. Lafleche
Introduction
89(5)
Voltage, current and power: interactive effects on gel electrophoresis
89(2)
Physical chemistry of agarose
91(3)
Preparation of agarose gels
94(14)
Buffer systems
94(1)
Dissolving agarose
95(2)
Casting an agarose gel
97(1)
Loading and running DNA in an agarose gel
98(2)
Detection of DNA in agarose gels
100(5)
Alkaline gel electrophoresis
105(1)
Drying an agarose gel
106(2)
Vertical agarose gels and fast-running horizontal gels
108(4)
Vertical gels
108(3)
Fast-running horizontal gels
111(1)
Electrophoresis of RNA
112(9)
Denaturing systems
112(1)
Preparation of RNA samples
113(2)
Electrophoresis of RNA
115(4)
References
119(2)
Recovery of DNA from Electrophoresis gels
121(8)
Paul Towner
Introduction
121(1)
Agarose gels
122(4)
Recovery of DNA from agarose gels
122(4)
Limitations on DNA recovery from agarose
126(1)
Polyacrylamide gels
126(3)
Recovery of DNA from polyacrylamide gels
127(1)
Acknowledgement
128(1)
References
128(1)
Construction of recombinant DNA molecules
129(22)
Richard Powell
Frank Gannon
Introduction
129(1)
Restriction enzyme digestions
130(7)
Preparation of vectors for molecular cloning experiments
137(5)
Phage λ
138(2)
Cosmids
140(1)
Plasmids
141(1)
Construction of recombinant molecules
142(7)
Ligation
142(3)
Linkers and adapters
145(2)
Tailing
147(2)
T/A cloning of PCR products
149(1)
Conclusion
149(2)
Acknowledgements
149(1)
References
150(1)
Generation and identification of recombinant clones
151(24)
T. A. Brown
Introduction
151(1)
Introduction of DNA into E. coli cells
151(10)
Uptake of DNA by chemically treated cells
152(5)
DNA uptake by electroporation
157(1)
In vitro packaging
158(3)
Plating out and recombinant selection
161(8)
Selection of plasmid vectors carrying antibiotic resistance genes
162(2)
Lac selection of plasmids
164(2)
Recombinant selection with M13 vectors
166(2)
Recombinant selection with λ vectors
168(1)
Vectors combining features of both plasmids and phages
169(6)
Cosmids
171(1)
Phagemids
171(2)
References
173(2)
Survey of cloning vectors for Escherichia coli
175(62)
T. A. Brown
Introduction
175(1)
E. coli as the host organism for recombinant DNA research
176(2)
Advantages of E. coli as a host organism
176(1)
E. coli is not an ideal host for gene cloning
177(1)
Plasmid vectors
178(19)
General properties of plasmid vectors
179(2)
Basic plasmid vectors
181(3)
Phagemids
184(2)
RNA expression vectors
186(2)
Protein expression vectors
188(3)
Cosmids
191(4)
Cloning vectors for PCR products
195(2)
Multipurpose vectors
197(1)
Bacteriophage λ vectors
197(7)
Practical considerations relevant to the use of λ vectors
198(3)
Examples of λ vectors
201(3)
Bacteriophage M13 vectors
204(5)
General properties of M13 vectors
204(2)
Acknowledgements
206(1)
References
206(3)
Appendices
A1 Important Escherichia coli strains
209(6)
A2 Recipes and general procedures
215(6)
A3 Restriction endonucleases
221(8)
A4 DNA and RNA modification enzymes
229(4)
A5 List of suppliers
233(4)
Index 237

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