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Tables of Contents for Molecular Cloning
Chapter/Section Title
Page #
Page Count
Preface
xxi
Acknowledgments
xxiii
On-line Edition
xxv
Quotation Credits
xxvii
Volume 1
Plasmids and Their Usefulness in Molecular Cloning
1
1
Introduction
Protocols
Introduction to Preparation of Plasmid DNA by Alkaline Lysis with SDS (Protocols 1--3)
31
1
Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation
32
3
Preparation of Plasmid DNA by Alkaline Lysis with SDS: Midipreparation
35
3
Preparation of Plasmid DNA by Alkaline Lysis with SDS: Maxipreparation
38
5
Introduction to Preparation of Plasmid DNA by Boiling Lysis (Protocols 4 and 5)
43
1
Preparation of Plasmid DNA by Small-scale Boiling Lysis
44
3
Preparation of Plasmid DNA by Large-scale Boiling Lysis
47
4
Preparation of Plasmid DNA: Toothpick Minipreparation
51
4
Preparation of Plasmid DNA by Lysis with SDS
55
4
Purification of Plasmid DNA by Precipitation with Polyethylene Glycol
59
3
Purification of Plasmid DNA by Chromatography
62
3
Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients: Continuous Gradients
65
4
Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients: Discontinuous Gradients
69
3
Removal of Ethidium Bromide from DNA by Extraction with Organic Solvents
72
3
Removal of Ethidium Bromide from DNA by Ion-exchange Chromatography
75
3
Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Centrifugation through NaCl
78
2
Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Chromatography through Sephacryl S-1000
80
2
Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Precipitation with Lithium Chloride
82
2
Directional Cloning into Plasmid Vectors
84
4
Attaching Adaptors to Protruding Termini
88
2
Blunt-ended Cloning into Plasmid Vectors
90
3
Dephosphorylation of Plasmid DNA
93
5
Addition of Synthetic Linkers to Blunt-ended DNA
98
5
Ligating Plasmid and Target DNAs in Low-melting-temperature Agarose
103
2
The Hanahan Method for Preparation and Transformation of Competent E. coli: High-efficiency Transformation
105
7
The Inoue Method for Preparation and Transformation of Competent E. coli: ``Ultra-competent'' Cells
112
4
Preparation and Transformation of Competent E. coli using Calcium Chloride
116
3
Transformation of E. coli by Electroporation
119
4
Screening Bacterial Colonies Using X-gal and IPTG: α-Complementation
123
2
Alternative Protocol: Direct Application of X-gal and IPTG to Agar Plates
125
1
Screening Bacterial Colonies by Hybridization: Small Numbers
126
3
Screening Bacterial Colonies by Hybridization: Intermediate Numbers
129
2
Alternative Protocol: Rapid Lysis of Colonies and Binding of DNA to Nylon Filters
131
1
Screening Bacterial Colonies by Hybridization: Large Numbers
132
3
Lysing Colonies and Binding of DNA to Filters
135
3
Hybridization of Bacterial DNA on Filters
138
5
Information Panels
Chloramphenicol
143
2
Kanamycins
145
1
pBR322
146
1
Tetracycline
147
1
Ampicillin and Carbenicillin
148
1
X-gal
149
1
α-Complementation
149
1
Ethidium Bromide
150
2
Condensing and Crowding Reagents
152
1
Purification of Plasmid DNA by PEG Precipitation
152
1
Lysozymes
153
1
Polyethylene Glycol
154
1
Cesium Chloride and Cesium Chloride Equilibrium Density Gradients
154
3
DNA Ligases
157
3
Adaptors
160
2
Electroporation
162
Bacteriophage λ and Its Vectors
1
1
Introduction
Protocols
Plating Bacteriophage λ
25
5
Additional Protocol: Plaque-Assay of Bacteriophages That Express β-Galactosidase
30
1
Additional Protocol: Macroplaques
31
1
Picking Bacteriophage λ Plaques
32
2
Preparing Stocks of Bacteriophage λ by Plate Lysis and Elution
34
3
Alternative Protocol: Preparing Stocks of Bacteriophage λ by Plate Lysis and Scraping
37
1
Preparing Stocks of Bacteriophage λ by Small-scale Liquid Culture
38
2
Large-scale Growth of Bacteriophage λ: Infection at Low Multiplicity
40
2
Alternative Protocol: Large-scale Growth of Bacteriophage λ: Infection at High Multiplicity
42
1
Precipitation of Bacteriophage λ Particles from Large-scale Lysates
43
2
Assaying the DNA Content of Bacteriophage λ Stocks and Lysates by Gel Electrophoresis
45
2
Purification of Bacteriophage λ Particles by Isopycnic Centrifugation through CsCl Gradients
47
4
Alternative Protocol: Purification of Bacteriophage λ Particles by Isophycnic Centrifugation through CsCl Equilibration Gradients
51
1
Purification of Bacteriophage λ Particles by Centrifugation through a Glycerol Step Gradient
52
2
Purification of Bacteriophage λ Particles by Pelleting/Centrifugation
54
2
Extraction of Bacteriophage λ DNA from Large-scale Cultures Using Proteinase K and SDS
56
3
Extraction of Bacteriophage λ DNA from Large-scale Cultures Using Formamide
59
2
Preparation of Bacteriophage λ DNA Cleaved with a Single Restriction Enzyme for Use as a Cloning Vector
61
3
Preparation of Bacteriophage λ DNA Cleaved with Two Restriction Enzymes for Use as a Cloning Vector
64
4
Alkaline Phosphatase Treatment of Bacteriophage λ Vector DNA
68
3
Purification of Bacteriophage λ Arms: Centrifugation through Sucrose Density Gradients
71
5
Partial Digestion of Eukaryotic DNA for Use in Genomic Libraries: Pilot Reactions
76
4
Partial Digestion of Eukaryotic DNA for Use in Genomic Libraries: Preparative Reactions
80
4
Ligation of Bacteriophage λ Arms to Fragments of Foreign Genomic DNA
84
3
Amplification of Genomic Libraries
87
3
Transfer of Bacteriophage DNA from Plaques to Filters
90
5
Alternative Protocol: Rapid Transfer of Plaques to Filters
95
1
Hybridization of Bacteriophage DNA on Filters
96
5
Rapid Analysis of Bacteriophage λ Isolates: Purification of λ DNA from Plate Lysates
101
4
Additional Protocol: Removing Polysaccharides by Precipitation with CTAB
105
1
Rapid Analysis of Bacteriophage λ Isolates: Purification of λ DNA from Liquid Cultures
106
3
Information Panels
Bacteriophages: Historical Perspective
109
1
Minimizing Damage to Large DNA Molecules
110
1
In Vitro Packaging
110
Working with Bacteriophage M13 Vectors
1
1
Introduction
Protocols
Plating Bacteriophage M13
17
3
Growing Bacteriophage M13 in Liquid Culture
20
3
Preparation of Double-stranded (Replicative Form) Bacteriophage M13 DNA
23
3
Preparation of Single-stranded Bacteriophage M13 DNA
26
4
Large-scale Preparation of Single-stranded and Double-stranded Bacteriophage M13 DNA
30
3
Cloning into Bacteriophage M13 Vectors
33
6
Analysis of Recombinant Bacteriophage M13 Clones
39
2
Alternative Protocol: Screening Bacteriophage M13 Plaques by Hybridization
41
1
Producing Single-stranded DNA with Phagemid Vectors
42
7
Information Panels
Growth Times
49
1
Polyethylene Glycol
49
Working with High-capacity Vectors
1
1
Introduction
Protocols
Construction of Genomic DNA Libraries in Cosmid Vectors
11
13
Screening an Unamplified Cosmid Library by Hybridization: Plating the Library onto Filters
24
3
Additional Protocol: Reducing Cross-hybridization
27
1
Amplification and Storage of a Cosmid Library: Amplification in Liquid Culture
28
3
Amplification and Storage of a Cosmid Library: Amplification on Filters
31
3
Alternative Protocol: Amplification on Plates
34
1
Working with Bacteriophage P1 and Its Cloning Systems
35
9
Additional Protocol: Purification of High-molecular-weight DNA by Drop Analysis
44
1
Alternative Protocol: Purification of High-molecular-weight Circular DNA by Chromatography on Qiagen Resin
45
1
Transferring P1 Clones between E. coli Hosts
46
2
Working with Bacterial Artificial Chromosomes
48
5
Isolation of BAC DNA from Small-scale Cultures
53
2
Isolation of BAC DNA from Large-scale Cultures
55
3
Working with Yeast Artificial Chromosomes
58
9
Growth of S. cerevisiae and Preparation of DNA
67
3
Small-scale Preparations of Yeast DNA
70
2
Analyzing Yeast Colonies by PCR
72
2
Isolating the Ends of Genomic DNA Fragments Cloned in High-capacity Vectors: Vectorette Polymerase Chain Reactions
74
8
Information Panels
Cre-loxP
82
4
Large-fragment Cloning Products and Services
86
Gel Electrophoresis of DNA and Pulsed-field Agarose Gel Electrophoresis
1
1
Introduction
Protocols
Agarose Gel Electrophoresis
4
10
Detection of DNA in Agarose Gels
14
4
Recovery of DNA from Agarose Gels: Electrophoresis onto DEAE-cellulose Membranes
18
5
Recovery of DNA from Agarose and Polyacrylamide Gels: Electroelution into Dialysis Bags
23
3
Purification of DNA Recovered from Agarose and Polyacrylamide Gas by Anion-exchange Chromatography
26
3
Recovery of DNA from Low-melting-temperature Agarose Gels: Organic Extraction
29
3
Alternative Protocol: Recovery of DNA from Agarose Gels Using Glass Beads
32
1
Recovery of DNA from Low-melting-temperature Agarose Gels: Enzymatic Digestion with Agarase
33
3
Alkaline Agarose Gel Electrophoresis
36
3
Additional Protocol: Autoradiography of Alkaline Agarose Gels
39
1
Neutral Polyacrylamide Gel Electrophoresis
40
7
Detection of DNA in Polyacrylamide Gels by Staining
47
2
Detection of DNA in Polyacrylamide Gels by Autoradiography
49
2
Isolation of DNA Fragments from Polyacrylamide Gels by the Crush and Soak Method
51
4
Introduction to Pulsed-field Gel Electrophoresis (Protocols 13--20)
55
6
Preparation of DNA for Pulsed-field Gel Electrophoresis: Isolation of DNA from Mammalian Cells and Tissues
61
4
Preparation of DNA for Pulsed-field Gel Electrophoresis: Isolation of Intact DNA from Yeast
65
3
Restriction Endonuclease Digestion of DNA in Agarose Plugs
68
3
Markers for Pulsed-field Gel Electrophoresis
71
3
Pulsed-field Gel Electrophoresis via Transverse Alternating Field Electrophoresis Gels
74
3
Alternative Protocol: Silver Staining PFGE Gels
77
2
Pulsed-field Gel Electrophoresis via Contour-clamped Homogeneous Electric Field Gels
79
4
Direct Retrieval of DNA Fragments from Pulsed-field Gels
83
3
Retrieval of DNA Fragments from Pulsed-field Gels following DNA Concentration
86
Preparation and Analysis of Eukaryotic Genomic DNA
1
1
Introduction
Protocols
Isolation of High-molecular-weight DNA from Mammalian Cells Using Proteinase K and Phenol
4
8
Additional Protocol: Estimating the Concentration of DNA by Fluorometry
12
1
Isolation of High-molecular-weight DNA from Mammalian Cells Using Formamide
13
3
Isolation of DNA from Mammalian Cells by Spooling
16
3
Isolation of DNA from Mammalian Cells Grown in 96-well Microtiter Plates
19
3
Additional Protocol: Optimizing Genomic DNA Isolation for PCR
22
1
Preparation of Genomic DNA from Mouse Tails and Other Small Samples
23
3
Alternative Protocol: Isolation of DNA from Mouse Tails without Extraction by Organic Solvents
26
1
Alternative Protocol: One-tube Isolation of DNA from Mouse Tails
26
1
Alternative Protocol: DNA Extraction from Paraffin Blocks
27
1
Rapid Isolation of Mammalian DNA
28
3
Rapid Isolation of Yeast DNA
31
2
Introduction to Southern Hybridization (Protocol 8--10)
33
6
Southern Blotting: Capillary Transfer of DNA to Membranes
39
8
Southern Blotting: Simultaneous Transfer of DNA from a Single Agarose Gel to Two Membranes
47
3
Southern Hybridization of Radiolabeled Probes to Nucleic Acids Immobilized on Membranes
50
7
Additional Protocol: Stripping Probes from Membranes
57
1
Additional Protocol: Hybridization at Low Stringency
58
1
Information Panels
Formamide and Its Uses in Molecular Cloning
59
2
Spooling DNA (Historical Footnote)
61
1
Rapid Hybridization Buffers
61
1
CTAB
62
Extraction, Purification, and Analysis of mRNA from Eukaryotic Cells
1
1
Introduction
Protocols
Purification of RNA from Cells and Tissues by Acid Phenol--Guanidinium Thiocyanate--Chloroform Extraction
4
5
A Single-step Method for the Simultaneous Preparation of DNA, RNA, and Protein from Cells and Tissues
9
4
Selection of Poly(A)+ RNA by Oligo(dT)-Cellulose Chromatography
13
5
Selection of Poly(A)+ RNA by Batch Chromatography
18
3
Introduction to Northern Hybridization (Protocols 5--9)
21
6
Separation of RNA According to Size: Electrophoresis of Glyoxylated RNA through Agarose Gels
27
4
Separation of RNA According to Size: Electrophoresis of RNA through Agarose Gels Containing Formaldehyde
31
4
Transfer and Fixation of Denatured RNA to Membranes
35
6
Alternative Protocol: Capillary Transfer by Downward Flow
41
1
Northern Hybridization
42
4
Dot and Slot Hybridization of Purified RNA
46
5
Mapping RNA with Nuclease S1
51
12
Ribonuclease Protection: Mapping RNA with Ribonuclease and Radiolabeled RNA Probes
63
12
Analysis of RNA by Primer Extension
75
7
Information Panels
How to Win the Battle with RNase
82
1
Inhibitors of RNases
83
1
Diethylpyrocarbonate
84
1
Guanidinium Salts
85
1
Nuclease S1
86
1
Exonuclease VII
86
1
Mung Bean Nuclease
87
1
Promoter Sequences Recognized by Bacteriophage-encoded RNA Polymerases
87
1
Actinomycin D
88
Index
I.1
Volume 2
In Vitro Amplification of DNA by the Polymerase Chain Reaction
1
1
Introduction
Protocols
The Basic Polymerase Chain Reaction
18
7
Purification of PCR Products in Preparation for Cloning
25
2
Removal of Oligonucleotides and Excess dNTPs from Amplified DNA by Ultrafiltration
27
3
Introduction to Cloning PCR Products (Protocols 4--7)
30
2
Blunt-end Cloning of PCR Products
32
3
Cloning PCR Products into T Vectors
35
2
Cloning PCR Products by Addition of Restriction Sites to the Termini of Amplified DNA
37
5
Genetic Engineering with PCR
42
4
Amplification of cDNA Generated by Reverse Transcription of mRNA (RT-PCR)
46
8
Rapid Amplification of 5' cDNA Ends (5'-RACE)
54
7
Rapid Amplification of 3' cDNA Ends (3'-RACE)
61
5
Mixed Oligonucleotide-primed Amplification of cDNA (MOPAC)
66
6
Rapid Characterization of DNAs Cloned in Prokaryotic Vectors
72
3
Additional Protocol: Screening Yeast Colonies by PCR
75
1
Additional Protocol: Screening Bacteriophage λ Libraries
76
1
Long PCR
77
4
Inverse PCR
81
5
Quantitative PCR
86
10
Differential Display-PCR (DD-PCR)
96
11
Information Panels
Multiplex PCR
107
1
Taq DNA Polymerase
108
2
Hot Start PCR
110
1
Ribonuclease H
111
1
Terminal Transferase
111
1
Touchdown PCR
112
1
Use of Inosine in Degenerate Pools of Oligonucleotides Used for PCR
113
1
Universal Primers
113
Preparation of Radiolabeled DNA and RNA Probes
1
1
Introduction
Protocols
Random Priming: Radiolabeling of Purified DNA Fragments by Extension of Random Oligonucleotides
4
5
Random Priming: Radiolabeling of DNA by Extension of Random Oligonucleotides in the Presence of Melted Agarose
9
3
Nick Translation: An Historical Note
12
2
Radiolabeling of DNA Probes by the Polymerase Chain Reaction
14
4
Additional Protocol: Asymmetric Probes
18
1
Synthesis of Single-stranded DNA Probes of Defined Length from Bacteriophage M13 Templates
19
6
Synthesis of Single-stranded DNA Probes of Heterogeneous Length from Bacteriophage M13 Templates
25
4
Synthesis of Single-stranded RNA Probes by In Vitro Transcription
29
7
Additional Protocol: Using PCR to Add Promoters for Bacteriophage-encoded RNA Polymerases to Fragments of DNA
36
2
Synthesis of cDNA Probes from mRNA Using Random Oligonucleotide Primers
38
3
Synthesis of Radiolabeled, Subtracted cDNA Probes Using Oligo(dT) as a Primer
41
5
Radiolabeling of Subtracted cDNA Probes by Random Oligonucleotide Extension
46
5
Labeling 3' Termini of Double-stranded DNA Using the Klenow Fragment of E. coli DNA Polymerase I
51
6
Labeling 3' Termini of Double-stranded DNA with Bacteriophage T4 DNA Polymerase
57
3
End Labeling Protruding 3' Termini of Double-stranded DNA with [α-32P]Cordycepin 5' Triphosphate or [α-32P]dideoxy ATP
60
2
Dephosphorylation of DNA Fragments with Alkaline Phosphatase
62
4
Phosphorylation of DNA Molecules with Protruding 5'-Hydroxyl Termini
66
4
Phosphorylation of DNA Molecules with Dephosphorylated Blunt Ends or Recessed 5' Termini
70
3
Phosphorylation of DNA Molecules with Protruding 5' Termini by the Exchange Reaction
73
3
Information Panels
Nonradioactive Labeling of Nucleic Acids
76
6
E. coli DNA Polymerase I and the Klenow Fragment
82
5
In Vitro Transcription Systems
87
2
Isolating Differentially Expressed cDNAs by Differential Screening and cloning
89
3
Alkaline Phosphatase
92
Working with Synthetic Oligonucleotide Probes
1
1
Introduction
Protocols
Purification of Synthetic Oligonucleotides by Polyacrylamide Gel Electrophoresis
11
6
Phosphorylating the 5' Termini of Oligonucleotides
17
3
Purification of Radiolabeled Oligonucleotides by Precipitation with Ethanol
20
2
Purification of Radiolabeled Oligonucleotides by Precipitation with Cetylpyridinium Bromide
22
3
Purification of Radiolabeled Oligonucleotides by Size-exclusion Chromatography
25
3
Purification of Radiolabeled Oligonucleotides by Chromatography on a Sep-Pak C18 Column
28
2
Labeling of Synthetic Oligonucleotides Using the Klenow Fragment of E. coli DNA Polymerase I
30
5
Hybridization of Oligonucleotide Probes in Aqueous Solutions: Washing in Buffers Containing Quaternary Ammonium Salts
35
3
Empirical Measurement of Melting Temperature
38
4
Information Panels
Oligonucleotide Synthesis
42
5
Melting Temperatures
47
1
Methods Used to Purify Synthetic Oligonucleotides
48
Preparation of cDNA Libraries and Gene Identification
1
1
Introduction
Protocols
Construction of cDNA Libraries
38
1
Synthesis of First-strand cDNA Catalyzed by Reverse Transcriptase
39
5
Second-strand Synthesis
44
5
Methylation of cDNA
49
3
Attachment of Linkers or Adaptors
52
5
Fractionation of cDNA by Gel Filtration through Sepharose CL-4B
57
3
Ligation of cDNA to Bacteriophage λ Arms
60
4
Alternative Protocol: Ligation of cDNA into a Plasmid Vector
64
1
Additional Protocol: Amplification of cDNA Libraries
65
3
Construction and Screening of Eukaryotic Expression Libraries
68
1
Construction of cDNA Libraries in Eukaryotic Expression Vectors
69
6
Screening cDNA Libraries Constructed in Eukaryotic Expression Vectors
75
5
Exon Trapping and Amplification
80
2
Construction of the Library
82
5
Electroporation of the Library into COS-7 Cells
87
2
Harvesting the mRNA
89
2
Reverse Transcriptase--PCR
91
5
Analysis of Clones
96
2
Direct Selection of cDNAs with Large Genomic DNA Clones
98
11
Information Panels
Commerical Kits for cDNA Synthesis and Library Construction
109
2
Mo-MLV Reverse Transcriptase
111
1
Homopolymeric Tailing
112
1
λgt10 and λgt11
113
1
Constructing cDNA Libraries from Small Numbers of Cells
114
1
In Vitro Packaging
115
1
COS Cells
116
1
Biotin
117
3
Magnetic Beads
120
3
Ligation-independent Cloning
123
DNA Sequencing
1
1
Introduction
Protocols
Generation of a Library of Randomly Overlapping DNA Inserts
10
13
Alternative Protocol: Preparation of Small Numbers of Single-stranded DNA Templates from Bacteriophage M13
23
1
Additional Protocol: Preparation of Dephosphorylated Blunt-ended Bacteriophage M13 Vector DNA for Shotgun Cloning
24
2
Preparing Denatured Templates for Sequencing by Dideoxy-mediated Chain Termination
26
4
Additional Protocol: Rapid Denaturation of Double-stranded DNA
30
1
Additional Protocol: Purification of Plasmid DNA from Small-scale Cultures by Precipitation with PEG
31
1
Dideoxy-mediated Sequencing Reactions Using Bacteriophage T7 DNA Polymerase (Sequenase)
32
8
Dideoxy-mediated Sequencing Reactions Using the Klenow Fragment of E. coli DNA Polymerase I and Single-stranded DNA Templates
40
5
Dideoxy-mediated Sequencing of DNA Using Taq DNA Polymerase
45
6
Cycle Sequencing: Dideoxy-mediated Sequencing Reactions Using PCR and End-labeled Primers
51
9
Additional Protocol: Cycle Sequencing Reactions Using PCR and Internal Labeling with [α-32P]dNTPs
60
1
Chemical Sequencing
61
9
Alternative Protocol: Rapid Maxam-Gilbert Sequencing
70
3
Additional Protocol: Preparation of End-labeled DNA for Chemical Sequencing
73
1
Preparation of Denaturing Polyacrylamide Gels
74
7
Preparation of Denaturing Polyacrylamide Gels Containing Formamide
81
2
Preparation of Electrolyte Gradient Gels
83
2
Loading and Running DNA-sequencing Gels
85
5
Autoradiography and Reading of Sequencing Gels
90
4
Infromation Panels
Automated DNA Sequencing
94
6
Microtiter Plates
100
1
The Klenow Fragment of E. coli DNA Polymerase I
101
2
Preparation of Stock Solutions of Oligonucleotide Primers for DNA Sequencing
103
1
Sequenase
104
2
Conventional Chain-termination Sequencing of PCR-amplified DNA
106
1
Preparation of Stock Solutions of dNTPs and ddNTPs for DNA Sequencing
107
1
Glycerol in DNA Sequencing Reactions
108
1
Compressions in DNA Sequencing Gels
109
2
7-deaza-dGTP
111
1
Dichlorodimethysilane
112
1
Reading an Autoradiograph
113
1
Elecrical Mobility of DNA
114
Mutagenesis
1
1
Introduction
Protocols
Preparation of Uracil-containing Single-stranded Bacteriophage M13 DNA
11
4
Oligonucleotide-directed Mutagenesis of Single-stranded DNA
15
4
In Vitro Mutagenesis Using Double-stranded DNA Templates: Selection of Mutants with Dpnl
19
7
Oligonucleotide-directed Mutagenesis by Elimination of a Unique Restriction Site (USE Mutagenesis)
26
5
Rapid and Efficient Site-directed Mutagenesis by the Single-tube Megaprimer PCR Method
31
5
Site-specific Mutagenesis by Overlap Extension
36
4
Screening Recombinant Clones for Site-directed Mutagenesis by Hybridization to Radiolabeled Oligonucleotides
40
7
Alternative Protocol: Screening Phagemid-containing Bacterial Colonies by Hybridization to Radiolabeled Oligonucleotides
47
1
Alternative Protocol: Detection of Defined Mutants by PCR
48
1
Detection of Mutations by Single-strand Conformational Polymorphism and Heteroduplex Analysis
49
8
Generation of Sets of Nested Deletion Mutants with Exonuclease III
57
5
Generation of Bidirectional Sets of Deletion Mutants by Digestion with BAL 31 Nuclease
62
6
Information Panels
BAL 31
68
4
Exonuclease III
72
3
Linker-scanning Mutagenesis
75
3
Random Mutagenesis
78
3
Alanine-scanning Mutagenesis
81
1
Mutagenic Oligonucleotides
82
2
Selecting against Wild-type DNA in Site-directed Mutagenesis
84
3
N6-methyladenine, Dam Methylase, and Methylation-sensitive Restriction Enzymes
87
2
Commercial Kits for Site-directed Mutagenesis
89
1
Glycerol
90
1
Mutation Detection
91
Screening Expression Libraries
1
1
Introduction
Protocols
Screening Expression Libraries Constructed in Bacteriophage λ Vectors
4
10
Screening Expression Libraries Constructed in Plasmid Vectors
14
9
Removal of Cross-reactive Antibodies from Antiserum: Pseudoscreening
23
2
Alternative Protocol: Adsorbing Antibodies with Lysates of Bacteriophage-infected Cells
25
1
Removal of Cross-reactive Antibodies from Antiserum: Incubation with E. coli Lysate
26
2
Removal of Cross-reactive Antibodies from Antiserum: Affinity Chromatography
28
3
Identifying DNA-binding Proteins in Bacteriophage λ Expression Libraries
31
6
Preparation of Lysates Containing Fusion Proteins Encoded by Bacteriophage λ Lysogens: Lysis of Bacterial Colonies
37
4
Preparation of Lysates Containing Fusion Proteins Encoded by Bacteriophage λ: Lytic Infections on Agar Plates
41
3
Preparation of Lysates Containing Fusion Proteins Encoded by Bacteriophage λ: Lytic Infections in Liquid Medium
44
3
Information Panels
Plasmid and Bacteriophage λ Expression Vectors
47
3
Using Antibodies in Immunological Screening
50
Index
I.1
Volume 3
Expression of Cloned Genes in Escherichia coli
1
1
Introduction
Protocols
Expression of Cloned Genes in E. coli Using IPTG-inducible Promoters
14
6
Expression of Cloned Genes in E. coli Using the Bacteriophage T7 Promoter
20
5
Expression of Cloned Genes in E. coli Using the Bacteriophage λ pL Promoter
25
5
Expression of Secreted Foreign Proteins Using the Alkaline Phosphatase Promoter (phoA) and Signal Sequence
30
5
Additional Protocol: Subcellular Localization of PhoA Fusion Proteins
35
1
Purification of Fusion Proteins by Affinity Chromatography on Glutathione Agarose
36
4
Purification of Maltose-binding Fusion Proteins by Affinity Chromatography on Amylose Resin
40
4
Purification of Histidine-tagged Proteins by Immobilized Ni2+ Absorption Chromatography
44
3
Alternative Protocol: Elution of Polyhistidine-tagged Proteins from Metal Affinity Columns Using Decreasing pH
47
1
Additional Protocol: Regeneration of NTA-Ni2+-Agarose
48
1
Purification of Expressed Proteins from Inclusion Bodies
49
4
Additional Protocol: Refolding Solubilized Proteins Recovered from Inclusion Bodies
53
2
Information Panels
Expression of Cloned Genes
55
1
E. coli Expression Systems
56
1
LacZ Fusions
57
3
Chaotropic Agents
60
Introducing Cloned Genes into Cultured Mammalian Cells
1
1
Introduction
Protocols
DNA Transfection Mediated by Lipofection
7
6
Additional Protocol: Histochemical Staining of Cell Monolayers for β-Galactosidase
13
1
Calcium-phosphate-mediated Transfection of Eukaryotic Cells with Plasmid DNAs
14
5
Alternative Protocol: High-efficiency Calcium-phosphate-mediated Transfection of Eukaryotic Cells with Plasmid DNAs
19
2
Clacium-phosphate-mediated Transfection of Cells with High-molecular-weight Genomic DNA
21
4
Alternative Protocol: Calcium-phosphate-mediated Transfection of Adherent Cells
25
1
Alternative Protocol: Calcium-phosphate-mediated Transfection of Cells Growing in Suspension
26
1
Transfection Mediated by DEAE-Dextran: High-efficiency Method
27
5
Alternative Protocol: Transfection Mediated by DEAE-Dextran: Increased Cell Viability
32
1
DNA Transfection by Electroporation
33
4
DNA Transfection by Biolistics
37
5
Additional Protocol: Histochemical Staining of Cell Monolayers or Tissue for β-Glucuronidase
42
1
DNA Transfection Using Polybrene
43
4
Information Panels
Cotransformation
47
1
Selective Agents for Stable Transformation
48
2
Lipofection
50
2
Transfection of Mammalian Cells with Calcium Phosphate--DNA Coprecipitates
52
1
Chloroquine Diphosphate
53
1
Electroporation
54
Analysis of Gene Expression in Cultured Mammalian Cells
1
1
Introduction
Protocols
Cis-acting Regions and Trans-acting Factors
Mapping Protein-binding Sites on DNA by DNase I Footprinting
4
8
Alternative Protocol: Mapping Protein-binding Sites on DNA by Hydroxyl Radical Footprinting
12
1
Gel Retardation Assays for DNA-binding Proteins
13
4
Additional Protocol: Supershift Assays
17
1
Additional Protocol: Competition Assays
17
1
Mapping DNase-I-hypersensitive Sites
18
5
Analysis of Primary Transcripts
Transcriptional Run-on Assays
23
7
Reporter Assays
Introduction to Reporter Assays: CAT, Luciferase, and β-galactosidase (Protocols 5--7)
30
3
Measurement of Chloramphenicol Acetyltransferase in Extracts of Mammalian Cells Using Thin-layer Chromatography
33
7
Alternative Protocol: Measurement of CAT by Extraction with Organic Solvents
40
1
Alternative Protocol: Measurement of CAT following Diffusion of Reaction Products into Scintillation Fluid
41
1
Assay for Luciferase in Extracts of Mammalian Cells
42
4
Alternative Protocol: Using a Scintillation Counter to Measure Luciferase
46
1
Alternative Protocol: Assay for Luciferase in Cells Growing in 96-well Plates
47
1
Assay for β-galactosidase in Extracts of Mammalian Cells
48
4
Inducible Systems
Tetracycline as Regulator of Inducible Gene Expression in Mammalian Cells
52
8
Stable Transfection of Fibroblasts with p-Tet-tTAk
60
5
Stable Transfection of Inducible tTA-expressing NIH-3T3 Cells with Tetracycline-regulated Target Genes
65
3
Analysis of Protein Expression in Transfected Cells
68
2
Alternative Protocol: Tetracycline-regulated Induction of Gene Expression in Transiently Transfected Cells Using the Autoregulatory tTA System
70
1
Ecdysone as Regulator of Inducible Gene Expression in Mammalian Cells
71
4
Information Panels
Footprinting DNA
75
3
Gel Retardation Assays
78
3
Baculoviruses and Baculovirus Expression Systems
81
3
Green Fluorescent Proteins
84
6
Epitope Tagging
90
4
Chloramphenicol Acetyltransferase
94
2
Luciferase
96
1
β-galactosidase
97
Protein Interaction Technologies
1
Introduction
Protocols
Two-hybrid and Other Two-component Systems
6
Characterization of a Bait-LexA Fusion Protein
17
Alternative Protocol: Assay of β-galactosidase Activity by Chloroform Overlay
28
Selecting an Interactor
30
Second Confirmation of Positive Interactions
38
Alternative Protocol: Rapid Screen for Interaction Trap Positives
46
Detection of Protein-Protein Interactions Using Far Western with GST Fusion Proteins
48
Additional Protocol: Refolding of Membrane-bound Proteins
53
Alternative Protocol: Detection of Protein-Protein Interactions with Anti-GST Antibodies
54
Detection of Protein-Protein Interactions Using the GST Fusion Protein Pulldown Technique
55
Identification of Associated Proteins by Coimmunoprecipitation
60
Probing Protein Interactions Using GFP and Fluorescence Resonance Energy Transfer
69
Labeling Proteins with Fluorescent Dyes
80
Cell Preparation for FLIM-FRET Analysis
84
Alternative Protocol: Preparation of Fixed Cells for FLIM-FRET Analysis
87
Alternative Protocol: Microinjection of Live Cells
88
FLIM-FRET Measurements
90
Analysis of Interacting Proteins with Surface Plasmon Resonance Spectroscopy Using BIAcore
96
Preparation of the Capture Surface and Test Binding
104
Kinetic Analysis of the Antibody-Antigen Interaction
108
Information Panels
Filamentous Phage Display
115
Genomics and the Interaction Trap
123
Interaction Trap and Related Technologies
125
Appendices
1 Preparation of Reagents and Buffers Used in Molecular Cloning
A1.1
2 Media
A2.1
3 Vectors and Bacterial Strains
A3.1
4 Enzymes Used in Molecular Cloning
A4.1
5 Inhibitors of Enzymes
A5.1
6 Nucleic Acids
A6.1
7 Codons and Amino Acids
A7.1
8 Commonly Used Techniques in Molecular Cloning
A8.1
9 Detection Systems
A9.1
10 DNA Array Technology
A10.1
11 Bioinformatics
A11.1
12 Cautions
A12.1
13 Suppliers
A13.1
14 Trademarks
A14.1
Appendix References
R1
Index
I.1