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Tables of Contents for Manipulation and Expression of Recombinant DNA
Chapter/Section Title
Page #
Page Count
Foreword
xiii
2
Acknowledgments
xv
2
Note to Instructors
xvii
INTRODUCTION Conceptual Outline for Experiments
1
8
I. PROCEDURES
1
1
II. LABORATORY SAFETY
2
2
III. MICROPIPETTES
4
5
PART I Manipulation of DNA
9
98
LAB SESSION 1 Culturing Escherichia coli for Plasmid Isolation
9
10
I. INTRODUCTION
9
1
II. ALPHA COMPLEMENTATION
9
3
III. LABORATORY EXERCISES
12
5
A. Preparation of Solid Media
12
1
B. Making Stab Cultures
13
1
C. Streaking out Plasmid Clones on LB/Carb Plates
13
2
D. Growth Curve of Escherichia coli
15
1
E. Growing Bacterial Suspension Cultures for Plasmid Preparations
16
1
REFERENCES
17
1
SOLUTIONS
17
2
LAB SESSION 2 Growth Curve of Escherichia coli
19
6
I. INTRODUCTION
19
1
II. PLASMID COPY CONTROL
20
1
III. LABORATORY EXERCISES
21
3
A. Examination of Clones
21
1
B. Escherichia coli Growth Curve
21
1
C. Preparation of Solutions for Large-Scale Plasmid DNA Preps
22
1
D. Inoculation of Cultures for Large-Scale Plasmid DNA Preps
23
1
SOLUTIONS
24
1
LAB SESSION 3A Large-Scale Preparation of Plasmid DNA: Alkaline Lysis
25
4
I. INTRODUCTION
25
1
II. LABORATORY EXERCISES
26
1
Alkaline Lysis of Large-Scale Cultures
26
1
REFERENCES
27
2
LAB SESSION 3B Purification of Plasmid DNA Using Columns
29
4
LABORATORY EXERCISES
29
2
REFERENCES
31
1
SOLUTIONS
31
2
LAB SESSION 3C Purification of Plasmid DNA Using Cesium Gradients
33
6
I. INTRODUCTION
33
1
II. LABORATORY EXERCISES
33
3
A. Beckman TLV-100 Table Top Ultracentrifuge with a Vertical Rotor
33
1
B. XL-90 Centrifuge
34
2
REFERENCES
36
1
SOLUTIONS
37
2
LAB SESSION 3D Precipitation of Plasmid DNA Using Polyethylene Glycol
39
2
I. INTRODUCTION
39
1
II. LABORATORY EXERCISES
39
1
REFERENCES
40
1
SOLUTIONS
40
1
LAB SESSION 4 Preparation of Vector DNA
41
10
I. INTRODUCTION
41
2
II. LABORATORY EXERCISES
43
5
A. Ethidium Bromide Spot Test
43
1
B. Preparation of HindIII-Cut, Phosphatased Vector
44
1
C. Gel Electrophoresis
45
1
D. Electrophoresis of pUR288
46
1
E. Shrimp Alkaline Phosphatase Treatment of pUR288
47
1
F. Overnight Cultures for Transformation-Competent Cells
48
1
SOLUTIONS
48
1
APPENDIX
49
2
LAB SESSION 5 Transformation of Escherichia coli
51
6
I. INTRODUCTION
51
1
II. LIGATION
51
1
III. LABORATORY EXERCISES
52
2
A. Preparation of Frozen, Transformation-Competent Cells
52
1
B. Test Ligations of pUR288
53
1
C. Electrophoresis of Ligation Products
53
1
D. Transformation
53
1
REFERENCES
54
1
SOLUTIONS
55
2
LAB SESSION 6 Preparation and Ligation of Insert DNA
57
6
I. INTRODUCTION
57
1
II. LIGATIONS
57
1
III. LABORATORY EXERCISES
58
4
A. Analysis of Vector Preparation
58
1
B. Isolation of myo-3 DNA from Agarose
59
2
C. Ligations
61
1
REFERENCES
62
1
SOLUTIONS
62
1
LAB SESSION 7A Transformation of Escherichia coli with Ligated DNA; Labeling a DNA Probe
63
4
I. INTRODUCTION
63
1
II. ELECTROPORATION
64
1
III. LABORATORY EXERCISES
64
1
A. Divalent Cation-Mediated Transformation
64
1
B. Electroporation
64
1
C. Electrophoresis of Ligation Reactions
65
1
D. Random Primed DNA Labeling with Digoxigenin-11-dUTP
65
1
REFERENCES
65
1
MATERIALS AND SOLUTIONS
66
1
LAB SESSION 7B Labeling DNA with Radioactive Nucleotides
67
4
I. INTRODUCTION
67
1
II. SAFETY PRECAUTIONS FOR WORKING WITH RADIOACTIVITY
68
1
III. LABORATORY EXERCISES
68
2
A. Random Primer Labeling of DNA Using [(32)P]dCTP
68
1
B. Using Spin Columns
69
1
MATERIALS AND SOLUTIONS
70
1
LAB SESSION 8 DNA Probe Quantitation and Replica Plating
71
4
I. INTRODUCTION
71
1
II. LABORATORY EXERCISES
71
2
A. Estimating the Yield of Digoxigenin-Labeled Probe
71
1
B. Making Replica Plates
72
1
MATERIALS AND SOLUTIONS
73
2
LAB SESSION 9 Colony Hybridization I: Working with a DNA Probe
75
8
I. INTRODUCTION
75
1
II. LABORATORY EXERCISES
76
3
A. Colony Hybridizations: Transfer of Colonies to Nylon Membranes
76
1
B. Prehybridization
77
1
C. Hybridization with the Digoxigenin-11-dUTP-Labeled Probe
77
1
D. Prehybridization for [(32)P]dCTP-Labeled DNA Probe
77
1
E. DNA/DNA Hybridization: Adding [(32)P]dCTP-Labeled DNA Probe
78
1
REFERENCES
79
1
SOLUTIONS
79
1
APPENDIX
80
3
LAB SESSION 10 Restriction Map of Plasmid p2D; Colony Hybridization II
83
6
I. INTRODUCTION
83
1
II. LABORATORY EXERCISES
84
2
A. Restriction Enzyme Reactions
84
1
B. Colony Hybridization with a DNA Probe: Washing Filters Probed with Digoxigenin-11-dUTP-Labeled myo-3 Fragment
84
1
C. Colony Hybridization with a DNA Probe: Washing Filters Probed with [(32)P]dCTP-Labeled myo-3 Fragment
85
1
D. Gel Electrophoresis of Restriction Digests
86
1
REFERENCES
86
1
MATERIALS AND SOLUTIONS
87
2
LAB SESSION 11 Screening Colonies with Monoclonal Antibodies
89
4
I. INTRODUCTION
89
1
II. LABORATORY EXERCISES
90
1
A. Colony Hybridizations: Monoclonal Antibody Probe (Day 1)
90
1
B. Set-up for Alkaline Lysis Minipreps
91
1
REFERENCES
91
1
SOLUTIONS
92
1
LAB SESSION 12 Isolation and Characterization of Miniprep DNA from Potential Transformants
93
8
I. INTRODUCTION
93
1
II. POLYMERASE CHAIN REACTION
94
1
III. LABORATORY EXERCISES
94
4
A. Colony Hybridizations: Monoclonal Antibody Probe (Day 2)
94
1
B. Completion of Alkaline Lysis Minipreps
95
1
C. Restriction Enzyme Analysis of Miniprep DNA
96
1
D. Polymerase Chain Reaction Screen for Recombinant Clones
97
1
REFERENCES
98
1
SOLUTIONS
99
1
APPENDIX Mapping Exercise: Analysis of Miniprep DNA Cut with Restriction Enzyme EcoRV
100
1
LAB SESSION 13 Titering Bacteriophage Lambda
101
6
I. INTRODUCTION
101
1
II. LABORATORY EXERCISES
101
2
A. Titering Bacteriophage Lambda
101
2
B. Analysis of Polymerase Chain Reactions
103
1
C. Overnight Cultures for Electrophoresis of Proteins
103
1
REFERENCES
103
1
SOLUTIONS
103
4
PART II Expression, Detection, and Purification of Recombinant Proteins from Bacteria
107
42
LAB SESSION 14 Expression of Fusion Protein in Bacterial Minipreps and Preparation of Sodium Dodecyl Sulfate-Polyacrylamide Gels
107
10
I. INTRODUCTION
107
1
II. LABORATORY EXERCISES
107
5
A. Growing Bacterial Suspension Cultures for Gel and Immunoblot Analysis
107
1
B. Pouring Stacking Gels for SDS-Polyacrylamide Slab Gels
108
1
C. Denaturing Protiens with Sodium Dodecyl Sulfate
108
1
D. Preparing Molecular Weight Standards
109
1
E. Loading Samples on the Gel
109
1
F. Electrophoresis
109
1
G. Stopping Electrophoresis
110
1
H. Staining the Gel
110
1
I. Drying the Gel
110
1
J. Inoculating Cultures for Large-Scale Purification
111
1
K. Calculations
111
1
SOLUTIONS
112
3
APPENDIX Pouring SDS-Polyacrylamide Gels
115
2
LAB SESSION 15 Extraction of Recombinant Protein from Escherichia coli
117
10
I. INTRODUCTION
117
1
II. LABORATORY EXERCISES
117
4
A. Growing Bacterial Suspension Cultures for Fusion Protein Purification
117
2
B. Harvesting IPTG-Induced Expression Vector Cultures
119
1
C. Breaking Open Bacterial Cells
119
1
D. Removing Insoluble Debris from the Crude Homogenate
120
1
E. Precipitating Protein with Ammonium Sulfate
120
1
F. Purifying Protein by Affinity Chromatography
120
1
REFERENCES
121
1
SOLUTIONS
122
5
Introduction to Lab Session 16: Quantitation of the Yield and Purity of Recombinant Beta-Galactosidase Fusion Proteins
LAB SESSION 16A Protein Determinations
127
6
I. INTRODUCTION
127
1
II. LABORATORY EXERCISES
127
3
A. Preparing Standard BSA Dilutions
127
1
B. Performing a Nitrocellulose Spot Test
128
1
C. Preparing BSA Standard Curve in Preparation for Bradford Assays
128
1
D. Assaying the Unknown: Subjecting Protein Samples to the Bradford Assay
129
1
E. Calculating Protein Concentrations
130
1
REFERENCES
130
1
SOLUTIONS
131
1
APPENDIX Sample Calculations of Protein Concentrations
131
2
LAB SESSION 16B Beta-Galactosidase Assays
133
6
I. INTRODUCTION
133
1
II. LABORATORY EXERCISES
134
3
A. Performing a Practice Beta-Galactosidase Assay
134
1
B. Performing the Beta-Galactosidase Assays with Fusion Proteins
134
1
C. Calculations of Specific Activity
134
2
D. Presenting Data and Calculations
136
1
REFERENCES
137
1
SOLUTIONS
137
1
APPENDIX Performing Protein and Enzyme Assays with an ELISA Plate Reader
138
1
LAB SESSION 17 Immunological Analysis of the Purified Fusion Proteins
139
6
I. INTRODUCTION
139
1
II. LABORATORY EXERCISES
139
3
A. Performing SDS-PAGE in Preparation for Immunoblotting
139
1
B. Electrophoretic Transfer of Proteins to Nitrocellulose
140
2
C. Staining the Blot with Protein Dye, Amido Black
142
1
REFERENCES
142
1
SOLUTIONS
142
3
LAB SESSION 18 Immunoblot Development
145
4
I. INTRODUCTION
145
1
II. LABORATORY EXERCISES
145
1
A. Incubating the Blot with Primary Antibody (Monoclonal Antibody 5-6)
145
1
B. Incubation with Goat Anti-Mouse Peroxidase
145
1
C. Chemiluminescent Peroxidase Reaction
146
1
III. QUESTIONS
146
1
MATERIALS AND SOLUTIONS
146
1
APPENDIX Colorimetric Detection of Peroxidase Activity
146
3
APPENDICES
149
52
1. EQUIPMENT AND SUPPLIES
149
4
2. PREP LIST
153
42
3. GLOSSARY
195
6
INDEX
201