Molecular Cloning: A Laboratory Manual, Volume 2

Front Cover
The first two editions of this manual have been mainstays of molecular biology for nearly twenty years, with an unrivalled reputation for reliability, accuracy, and clarity. In this new edition, authors Joseph Sambrook and David Russell have completely updated the book, revising every protocol and adding a mass of new material, to broaden its scope and maintain its unbeatable value for studies in genetics, molecular cell biology, developmental biology, microbiology, neuroscience, and immunology. Handsomely redesigned and presented in new bindings of proven durability, this three-volume work is essential for everyone using today's biomolecular techniques. The opening chapters describe essential techniques, some well-established, some new, that are used every day in the best laboratories for isolating, analyzing and cloning DNA molecules, both large and small. These are followed by chapters on cDNA cloning and exon trapping, amplification of DNA, generation and use of nucleic acid probes, mutagenesis, and DNA sequencing. The concluding chapters deal with methods to screen expression libraries, express cloned genes in both prokaryotes and eukaryotic cells, analyze transcripts and proteins, and detect protein-protein interactions. The Appendix is a compendium of reagents, vectors, media, technical suppliers, kits, electronic resources and other essential information. As in earlier editions, this is the only manual that explains how to achieve success in cloning and provides a wealth of information about why techniques work, how they were first developed, and how they have evolved.

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Contents

Chapter 8
8
Chapter 12
12
Preparing Denatured Doublestranded DNA Templates for Sequencing by 12 26
26
Dideoxymediated Sequencing Reactions Using Bacteriophage T7 DNA Polymerase 12 32
32
Agarose Gel Electrophoresis 5 4
57
Chemical Sequencing 12 61
61
INFORMATION PANELS
94
In Vitro Amplification of DNA by the Polymerase
107

INFORMATION PANELS
107
INFORMATION PANELS
109
Chapter 9
7
Radiolabeling of DNA Probes by the Polymerase Chain Reaction 9 14
14
Chapter 15
15
Synthesis of Singlestranded RNA Probes by In Vitro Transcription 9 29
29
Phosphorylation of DNA Molecules with Protruding 5 Termini by the Exchange 9 73
73
INFORMATION PANELS
77
Chapter 10
10
Phosphorylating the 5 Termini of Oligonucleotides 10 17
17
Purification of Radiolabeled Oligonucleotides by Sizeexclusion Chromatography 10 25
25
Washing in Buffers 10 35
35
INFORMATION PANELS
43
1
1
Chapter 3
3
Chapter 5
5
Construction of cDNA Libraries 11 38
11
PROTOCOLS
13
Volume 2
57
Exon Trapping and Amplification 11 80
79
INFORMATION PANELS
98
INFORMATION PANELS
1
Chapter 7
7
Detection of DNA in Agarose Gels 5 14
117
INTRODUCTION
1
Electrophoresis onto DEAEcellulose 5 18
5
End Labeling Protruding 3 Termini of Doublestranded DNA with Q32PCordycepin 9 60
9
Chapter 13
13
Expression of Cloned Genes in Escherichia coli 15 1
15
Sitespecific Mutagenesis by Overlap Extension 13 36
36
Generation of Sets of Nested Deletion Mutants with Exonuclease III 13 57
57
INFORMATION PANELS
61
Digestion with Agarase
62
Chapter 2
2
Chapter 14
14
Chapter 16
16
PROTOCOLS
37
Chapter 4
4
Extraction Purification and Analysis of mRNA from 7 1
7
INFORMATION PANELS
11
1
1
Alkaline Agarose Gel Electrophoresis 5 36
5
Introduction to Pulsedfield Gel Electrophoresis Protocols 1320 5 55
13
Isolation of Intact DNA 5 65
34
Restriction Endonuclease Digestion of DNA in Agarose Plugs 5 68
41
Copyright

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Popular passages

Page 87 - Melton, DA, Krieg, PA, Rebagliati, MR, Maniatis, T., Zinn, K., and Green, MR (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12, 7035-7056.
Page 29 - Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Page 84 - Eldeiry, WS, Tokino, T., Velculescu. VE, Levy, DB, Parsons. R., Trent, JM, Lin, D., Mercer, WE, Kinzler, KW, and Vogelstein, B.
Page 88 - Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase.
Page 83 - Campbell, AP, Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, ED, and Siebert, PD (1996).
Page 120 - Gilliland. G., Perrin. S., Blanchard. K., and Bunn, HF (1990) Analysis of cytokine mRNA and DNA detection and quantitation by competitive polymerase chain reaction.
Page 120 - Frohman, MA, Dush, MK, and Martin, GR (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 8998-9002. 2. Edwards, JBDM, Delort, J., and Mallet, J. (1991) Oligodeoxyribonucleotide ligation to single-stranded cDNAs: A new tool for cloning 5' ends of mRNAs and for constructing cDNA libraries by in vitro amplification.
Page 83 - DV, Angerer, LM, and Angerer, RC (1984) Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev. Biol. 101, 485-502.
Page 97 - Birnboim, HC and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA.
Page 84 - A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.

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