Molecular Biology Techniques (eBook)

Front Cover 1
Molecular Biology Techniques: A Classroom Laboratory Manual 4
Copyright Page 5
Contents 6
Preface 12
About the Authors 14
Acknowledgements 16
Note to Instructors 18
Instrumentation 20
Nomenclature 22
Introduction 24
Conceptual Outline for Experiments 24
Experimental Procedures 24
Laboratory Safety 26
General Operating Procedures 27
1 – Manipulation of DNA 28
Reference 29
Lab Session 1. Getting Oriented: Practicing with Micropipettes 30
Station Checklist 30
Micropipetting 31
Micropipetting Self-Test 33
Laboratory Exercise: BSA Serial Dilutions and Nitrocellulose Spot Test 34
Preparing BSA Dilutions 34
Performing a Nitrocellulose Spot Test 34
Discussion Questions 36
Lab Session 2. Purification and Digestion of Plasmid (Vector) DNA 38
Introduction to Plasmid Purification 38
Alkaline Lysis 38
Silica Adsorption 39
DNA Quantification 39
Introduction to Expression Vectors 39
Principles of Gene Expression 40
Expression Vectors 40
Orientation and Reading Frame 41
Orientation 41
Reading Frame 42
Laboratory Exercises 43
Alkaline Lysis and Silica Adsorption Protocol 43
DNA Quantification 45
Restriction Digestion of Expression Vector DNA pET-41a, a GST Fusion Protein Vector 46
Discussion Questions 47
Reference 47
Lab Session 3. PCR Amplification of egfp and Completion of Vector Preparation 48
Introduction 48
What is the Polymerase Chain Reaction (PCR)? 48
Why Clone by PCR? 50
TA Cloning 50
PCR Cloning by Incorporation of Restriction Sites 50
Cloning Synthetic Genes 51
Laboratory Exercises 52
PCR Amplification of egfp from the pEGFP-N1 Plasmid 52
PCR Protocol 52
Clean-up of Digested pET-41a Vector 52
Agarose Gel Electrophoresis 54
Discussion Questions 56
References 56
Lab Session 4. Preparation of Insert DNA (egfp) PCR Product 58
Check PCR Reactions on an Agarose Gel 58
Spin Column Cleanup of PCR Product 58
Quantification of egfp PCR Product 58
Restriction Digestion of egfp PCR Product 59
Removing Enzymes and Cleaning Digested DNA Using a Spin Column 59
Discussion Questions 60
Lab Session 5. DNA Ligation and Transformation of Escherichia coli 62
Introduction 62
Ligation 62
Transformation 63
Laboratory Exercises 64
Ligations and Ligation Controls 64
Divalent Cation-Mediated Transformation 66
Electrophoresis of Ligation Reactions 66
Discussion Questions 67
Reference 67
2 – Screening Transformants 68
Lab Session 6. Colony Hybridization 70
Lab Session 6A. Interim Laboratory Session 71
Introduction 71
Laboratory Exercises 71
Counting Transformants and Replica Plating 71
Replica Plating 71
Lab Session 6B. Colony Hybridization: Monoclonal Antibody Probe 73
Introduction 73
Laboratory Exercises 74
Colony Hybridization with an a-EGFP Monoclonal Antibody Probe: Part 1 74
Lab Session 6. Discussion Questions 76
Lab Session 7. Characterization of Recombinant Clones: Part 1 78
Lab Session 7A. Completion of Colony Hybridization with a Monoclonal Antibody Probe 79
Introduction 79
Laboratory Exercise 79
Colony Hybridization with an a-EGFP Monoclonal Antibody Probe: Part 2 79
Lab Session 7B. PCR Screening 81
Introduction 81
Laboratory Exercise 81
Polymerase Chain Reaction Screen for Recombinant Clones 81
Lab Session 7C. Prepare Fresh Replica Plate 84
Lab Session 7. Discussion Questions 84
Lab Session 8. Characterization of Recombinant Clones: Part 2 86
Lab Session 8A. Interim Laboratory Session 87
Laboratory Exercise 87
Inoculate Cultures for Minipreps 87
Lab Session 8B. Analysis of PCR Screen Results 88
Introduction 88
Laboratory Exercise 88
Gel Electrophoresis and Analysis of PCR Samples from Last Week 88
Lab Session 8C. Isolation of Miniprep DNA from Potential Transformants 89
Introduction 89
Laboratory Exercise 90
Isolation of Miniprep DNA from Potential Transformants 90
Plasmid DNA Purification Using the QIAprep Spin Miniprep Kit and a Microcentrifuge 90
Lab Session 8D. Visualization of Green Fluorescent Protein: Part 1 92
Introduction 92
Laboratory Exercise 92
Green Fluorescence Assay and Preparation of a Fresh Master Plate 92
Lab Session 8. Discussion Questions 93
Lab Session 9. Characterization of Recombinant Clones: Part 3 94
Lab Session 9A. Characterization of Miniprep DNA from Potential Transformants (Restriction Enzyme Analysis of Putative Transformants) 95
Introduction 95
Laboratory Exercise 95
Restriction Enzyme Analysis of Miniprep DNA 95
Lab Session 9B. Visualization of Green Fluorescent Protein: Part 2 97
Introduction 97
Laboratory Exercise 97
Visualization of Clones Expressing the Enhanced Green Fluorescent Protein on IPTG Plates 97
Lab Session 9C. Computational Analysis of DNA Sequence from a Positive Clone: Part 1 99
Introduction 99
Laboratory Exercise 102
References 103
Lab Session 9. Discussion Questions 103
Lab Session 10. Computational Analysis of DNA Sequence from a Positive Clone: Part 2 104
Introduction 104
Laboratory Exercise 108
Discussion Questions 112
Reference 112
3 – Expression, Detection and Purification of Recombinant Proteins from Bacteria 114
Lab Session 11. Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot: Part 1 116
Lab Session 11A. Interim Laboratory Session 117
Laboratory Exercise 117
Inoculate Cultures for SDS-PAGE 117
Lab Session 11B. Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot 118
Introduction 118
Laboratory Exercise 120
SDS-PAGE and Western Blot: Part 1 120
Reference 124
Lab Session 11. Discussion Questions 124
Lab Session 12. Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot: Part 2 126
Introduction 126
Laboratory Exercises 126
SDS-PAGE and Western Blot: Part 2 126
Replica Plate Positive Clone 128
Discussion Questions 128
Lab Session 13. Extraction of Recombinant Protein from Escherichia coli Using a Glutathione Affinity Column 130
Lab Session 13A. Interim Laboratory Session 131
Laboratory Exercise 131
Inoculate Cultures for Protein Purification 131
Lab Session 13B. Extraction of Recombinant Protein from Escherichia coli and Purification Using a Glutathione Affinity Column 132
Introduction 132
Laboratory Exercises 135
Growing Bacterial Suspension Cultures for Fusion Protein Purification 135
Harvesting IPTG-Induced Cultures 135
Breaking Open Bacterial Cells 135
Removing Insoluble Debris from the Crude Homogenate 136
Purifying Protein by Affinity Chromatography 136
Lab Session 13. Discussion Questions 138
Lab Session 14. Analysis of Purification Fractions 140
Lab Session 14A. Analysis of Purification Fractions 141
Introduction 141
Laboratory Exercises 142
SDS-PAGE of Purified Fusion Protein 142
Fluorescence Analysis of Affinity Purification 143
Lab Session 14B. Replica Plate Positive Clone 147
Lab Session 14. Discussion Questions 147
4 – Analysis of mRNA Levels 148
Challenges of Working with RNA 149
References 150
Lab Session 15. Total RNA Purification 152
Lab Session 15A. Interim Laboratory Session 153
Laboratory Exercise 153
Inoculate Cultures for RNA Purification 153
Lab Session 15B. Total RNA Purification 154
Introduction 154
Laboratory Exercises 156
Purification of Total RNA 156
DNase Digestion 158
Quantification of RNA 158
References 159
Lab Session 15. Discussion Questions 159
Lab Session 16. Analysis of gst::egfp mRNA Levels by RT-qPCR: Part 1 160
Introduction 160
Reverse Transcription 161
Quantitative PCR 162
Laboratory Exercises 164
Reverse Transcription 164
Quantitative PCR (qPCR) 165
Discussion Questions 167
Reference 167
Lab Session 17. Analysis of gst::egfp mRNA Levels by RT-qPCR: Part 2 168
Introduction 168
Laboratory Exercise 171
Relative Quantification of gst::egfp Levels 171
References 173
Discussion Questions 173
Lab Session 18. Analysis of gst::egfp mRNA Levels by Semi-Quantitative RT-PCR: Part 1 174
Introduction 174
Laboratory Exercises 175
Reverse Transcription (RT) 175
Semi-Quantitative PCR 175
Discussion Questions 178
Lab Session 19. Analysis of gst::egfp mRNA Levels by Semi-Quantitative RT-PCR: Part 2 180
Introduction 180
Laboratory Exercises 180
Agarose Gel Electrophoresis 180
Quantification 181
Discussion Questions 182
Reference 182
Appendix 1: Equipment 184
Shared Equipment 184
Appendix 2: Prep List 186
Notes to Prep Staff 186
Plasmids and E. coli Host Strains 186
Antibiotics 186
Aliquots 187
Bacterial Waste 187
Autoclaving 187
General Lab Preparation 188
Students 188
Supplies and Reagents 188
Supplies and Reagents for General Use 188
Recipes for General Use 189
LAB SESSION 1 190
BSA Serial Dilutions and Nitrocellulose Spot Test 190
Recipes 190
LAB SESSION 2 191
Purification and Digestion of Plasmid (Vector) DNA 191
LAB SESSION 3 192
PCR Amplification of egfp from pEGFP-N1 Clean-up and Visualization of Digested pET-41a Vector
Recipes 193
LAB SESSION 4 193
Preparation of Insert DNA (egfp) PCR Product 193
LAB SESSION 5 193
DNA Ligation and Transformation of Escherichia coli 193
LAB SESSION 6 194
Interim Lab 194
LAB SESSION 6 195
Colony Hybridization: Monoclonal Antibody Probe 195
Recipes 195
LAB SESSION 7 196
Characterization of Recombinant Clones 196
Recipes 197
Chloronaphthol Stock Solution 197
Peroxide Stain 197
LAB SESSION 8 197
Interim Lab 197
LAB SESSION 8 198
Characterization of Recombinant Clones: Part 2 198
LAB SESSION 9 198
Characterization of Recombinant Clones: Part 3 198
LAB SESSION 10 199
Computational Analysis of DNA Sequence from a Positive Clone: Part 2 199
LAB SESSION 11 199
Interim Lab 199
LAB SESSION 11 199
Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot: Part 1 199
Recipes 200
LAB SESSION 12 201
Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot: Part 2 201
Recipes 202
TBS (Tris-buffered Saline, pH 7.6) 202
LAB SESSION 13 202
Interim Lab 202
LAB SESSION 13 202
Extraction of Recombinant Protein from Escherichia coli Using a Glutathione Affinity Column 202
Recipes 203
LAB SESSION 14 203
Analysis of Purification Fractions 203
Recipes 204
LAB SESSION 15 204
Interim Lab 204
LAB SESSION 15 204
Total RNA Purification 204
Recipes 205
LAB SESSION 16 206
Analysis of gst::egfp mRNA Levels by RT-qPCR: Part 1 206
LAB SESSION 17 206
Analysis of gst::egfp mRNA Levels by RT-qPCR: Part 2 206
LAB SESSION 18 206
Analysis of gst::egfp mRNA Levels by Semi-Quantitative RT-PCR: Part 1 206
LAB SESSION 19 207
Analysis of gst::egfp mRNA Levels by Semi-Quantitative RT-PCR: Part 2 207
Appendix 3: Preparation of Competent E. coli Cells 208
Introduction 208
Protocol 208
Preparation of Chemically Competent Cells by Calcium Chloride Treatment 208
Transformation Control 209
Appendix 4: Pre-Lab Questions 212
LAB SESSION 1 212
LAB SESSION 2 212
LAB SESSION 3 213
LAB SESSION 4 213
LAB SESSION 5 214
LAB SESSION 6 214
LAB SESSION 7 215
LAB SESSION 8 215
LAB SESSION 9 216
LAB SESSION 10 216
LAB SESSION 11 217
LAB SESSION 12 218
LAB SESSION 13 218
LAB SESSION 14 219
LAB SESSION 15 219
LAB SESSION 16 220
LAB SESSION 17 220
LAB SESSION 18 221
LAB SESSION 19 222
Index 224