Miller / Witherow / Carson Molecular Biology Techniques

A Classroom Laboratory Manual
3. Auflage 2011
ISBN: 978-0-12-385545-9
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: Adobe DRM (»Systemvoraussetzungen)

E-Book, Englisch, 232 Seiten

This manual is an indispensable tool for introducing advanced undergraduates and beginning graduate students to the techniques of recombinant DNA technology, or gene cloning and expression. The techniques used in basic research and biotechnology laboratories are covered in detail. Students gain hands-on experience from start to finish in subcloning a gene into an expression vector, through purification of the recombinant protein. The third edition has been completely re-written, with new laboratory exercises and all new illustrations and text, designed for a typical 15-week semester, rather than a 4-week intensive course. The 'project approach to experiments was maintained: students still follow a cloning project through to completion, culminating in the purification of recombinant protein. It takes advantage of the enhanced green fluorescent protein - students can actually visualize positive clones following IPTG induction. - Cover basic concepts and techniques used in molecular biology research labs - Student-tested labs proven successful in a real classroom laboratories - Exercises simulate a cloning project that would be performed in a real research lab - 'Project' approach to experiments gives students an overview of the entire process - Prep-list appendix contains necessary recipes and catalog numbers, providing staff with detailed instructions

Dr. Heather Miller is an Assistant Professor of Biochemistry in the Chemistry Department at High Point University (High Point, NC). She graduated from Clarion University of Pennsylvania (Clarion, PA) with a B.S. in Molecular Biology/Biotechnology, and from Duke University (Durham, NC) with a Ph.D. in Molecular Genetics and Microbiology. She completed a teaching postdoctoral position in the Biotechnology Program at North Carolina State University (Raleigh, NC). Her area of scientific expertise is RNA biology. Her research focuses on HIV-1 gene expression and the coupling of transcription and splicing in humans. She has taught at the college level for nine years, and is engaged in the scholarship of teaching and learning.

Autoren/Hrsg.

Miller, Heather B.

Witherow, D. Scott

Carson, Sue

Inhaltsverzeichnis

1;Front Cover;1
2;Molecular Biology Techniques: A Classroom Laboratory Manual;4
3;Copyright Page;5
4;Contents;6
5;Preface;12
6;About the Authors;14
7;Acknowledgements;16
8;Note to Instructors;18
9;Instrumentation;20
10;Nomenclature;22
11;Introduction;24
11.1;Conceptual Outline for Experiments;24
11.2;Experimental Procedures;24
11.3;Laboratory Safety;26
11.4;General Operating Procedures;27
12;1 – Manipulation of DNA;28
12.1;Reference;29
12.2;Lab Session 1. Getting Oriented: Practicing with Micropipettes;30
12.2.1;Station Checklist;30
12.2.2;Micropipetting;31
12.2.2.1;Micropipetting Self-Test;33
12.2.3;Laboratory Exercise: BSA Serial Dilutions and Nitrocellulose Spot Test;34
12.2.3.1;Preparing BSA Dilutions;34
12.2.3.2;Performing a Nitrocellulose Spot Test;34
12.2.4;Discussion Questions;36
12.3;Lab Session 2. Purification and Digestion of Plasmid (Vector) DNA;38
12.3.1;Introduction to Plasmid Purification;38
12.3.2;Alkaline Lysis;38
12.3.2.1;Silica Adsorption;39
12.3.2.2;DNA Quantification;39
12.3.3;Introduction to Expression Vectors;39
12.3.4;Principles of Gene Expression;40
12.3.4.1;Expression Vectors;40
12.3.5;Orientation and Reading Frame;41
12.3.5.1;Orientation;41
12.3.5.2;Reading Frame;42
12.3.6;Laboratory Exercises;43
12.3.6.1;Alkaline Lysis and Silica Adsorption Protocol;43
12.3.6.2;DNA Quantification;45
12.3.6.3;Restriction Digestion of Expression Vector DNA pET-41a, a GST Fusion Protein Vector;46
12.3.7;Discussion Questions;47
12.3.8;Reference;47
12.4;Lab Session 3. PCR Amplification of egfp and Completion of Vector Preparation;48
12.4.1;Introduction;48
12.4.2;What is the Polymerase Chain Reaction (PCR)?;48
12.4.3;Why Clone by PCR?;50
12.4.3.1;TA Cloning;50
12.4.3.2;PCR Cloning by Incorporation of Restriction Sites;50
12.4.4;Cloning Synthetic Genes;51
12.4.5;Laboratory Exercises;52
12.4.5.1;PCR Amplification of egfp from the pEGFP-N1 Plasmid;52
12.4.5.2;PCR Protocol;52
12.4.5.3;Clean-up of Digested pET-41a Vector;52
12.4.5.4;Agarose Gel Electrophoresis;54
12.4.6;Discussion Questions;56
12.4.7;References;56
12.5;Lab Session 4. Preparation of Insert DNA (egfp) PCR Product;58
12.5.1;Check PCR Reactions on an Agarose Gel;58
12.5.2;Spin Column Cleanup of PCR Product;58
12.5.3;Quantification of egfp PCR Product;58
12.5.4;Restriction Digestion of egfp PCR Product;59
12.5.5;Removing Enzymes and Cleaning Digested DNA Using a Spin Column;59
12.5.6;Discussion Questions;60
12.6;Lab Session 5. DNA Ligation and Transformation of Escherichia coli;62
12.6.1;Introduction;62
12.6.1.1;Ligation;62
12.6.1.2;Transformation;63
12.6.2;Laboratory Exercises;64
12.6.2.1;Ligations and Ligation Controls;64
12.6.2.2;Divalent Cation-Mediated Transformation;66
12.6.2.3;Electrophoresis of Ligation Reactions;66
12.6.3;Discussion Questions;67
12.6.4;Reference;67
13;2 – Screening Transformants;68
13.1;Lab Session 6. Colony Hybridization;70
13.2;Lab Session 6A. Interim Laboratory Session;71
13.2.1;Introduction;71
13.2.2;Laboratory Exercises;71
13.2.2.1;Counting Transformants and Replica Plating;71
13.2.2.2;Replica Plating;71
13.3;Lab Session 6B. Colony Hybridization: Monoclonal Antibody Probe;73
13.3.1;Introduction;73
13.3.2;Laboratory Exercises;74
13.3.2.1;Colony Hybridization with an a-EGFP Monoclonal Antibody Probe: Part 1;74
13.4;Lab Session 6. Discussion Questions;76
13.5;Lab Session 7. Characterization of Recombinant Clones: Part 1;78
13.6;Lab Session 7A. Completion of Colony Hybridization with a Monoclonal Antibody Probe;79
13.6.1;Introduction;79
13.6.2;Laboratory Exercise;79
13.6.2.1;Colony Hybridization with an a-EGFP Monoclonal Antibody Probe: Part 2;79
13.7;Lab Session 7B. PCR Screening;81
13.7.1;Introduction;81
13.7.2;Laboratory Exercise;81
13.7.2.1;Polymerase Chain Reaction Screen for Recombinant Clones;81
13.8;Lab Session 7C. Prepare Fresh Replica Plate;84
13.9;Lab Session 7. Discussion Questions;84
13.10;Lab Session 8. Characterization of Recombinant Clones: Part 2;86
13.11;Lab Session 8A. Interim Laboratory Session;87
13.11.1;Laboratory Exercise;87
13.11.1.1;Inoculate Cultures for Minipreps;87
13.12;Lab Session 8B. Analysis of PCR Screen Results;88
13.12.1;Introduction;88
13.12.2;Laboratory Exercise;88
13.12.2.1;Gel Electrophoresis and Analysis of PCR Samples from Last Week;88
13.13;Lab Session 8C. Isolation of Miniprep DNA from Potential Transformants;89
13.13.1;Introduction;89
13.13.2;Laboratory Exercise;90
13.13.2.1;Isolation of Miniprep DNA from Potential Transformants;90
13.13.2.2;Plasmid DNA Purification Using the QIAprep Spin Miniprep Kit and a Microcentrifuge;90
13.14;Lab Session 8D. Visualization of Green Fluorescent Protein: Part 1;92
13.14.1;Introduction;92
13.14.2;Laboratory Exercise;92
13.14.2.1;Green Fluorescence Assay and Preparation of a Fresh Master Plate;92
13.15;Lab Session 8. Discussion Questions;93
13.16;Lab Session 9. Characterization of Recombinant Clones: Part 3;94
13.17;Lab Session 9A. Characterization of Miniprep DNA from Potential Transformants (Restriction Enzyme Analysis of Putative Transformants);95
13.17.1;Introduction;95
13.17.2;Laboratory Exercise;95
13.17.2.1;Restriction Enzyme Analysis of Miniprep DNA;95
13.18;Lab Session 9B. Visualization of Green Fluorescent Protein: Part 2;97
13.18.1;Introduction;97
13.18.2;Laboratory Exercise;97
13.18.2.1;Visualization of Clones Expressing the Enhanced Green Fluorescent Protein on IPTG Plates;97
13.19;Lab Session 9C. Computational Analysis of DNA Sequence from a Positive Clone: Part 1;99
13.19.1;Introduction;99
13.19.2;Laboratory Exercise;102
13.19.3;References;103
13.20;Lab Session 9. Discussion Questions;103
13.21;Lab Session 10. Computational Analysis of DNA Sequence from a Positive Clone: Part 2;104
13.21.1;Introduction;104
13.21.2;Laboratory Exercise;108
13.21.3;Discussion Questions;112
13.21.4;Reference;112
14;3 – Expression, Detection and Purification of Recombinant Proteins from Bacteria;114
14.1;Lab Session 11. Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot: Part 1;116
14.2;Lab Session 11A. Interim Laboratory Session;117
14.2.1;Laboratory Exercise;117
14.2.1.1;Inoculate Cultures for SDS-PAGE;117
14.3;Lab Session 11B. Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot;118
14.3.1;Introduction;118
14.3.2;Laboratory Exercise;120
14.3.2.1;SDS-PAGE and Western Blot: Part 1;120
14.3.3;Reference;124
14.4;Lab Session 11. Discussion Questions;124
14.5;Lab Session 12. Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot: Part 2;126
14.5.1;Introduction;126
14.5.2;Laboratory Exercises;126
14.5.2.1;SDS-PAGE and Western Blot: Part 2;126
14.5.2.2;Replica Plate Positive Clone;128
14.5.3;Discussion Questions;128
14.6;Lab Session 13. Extraction of Recombinant Protein from Escherichia coli Using a Glutathione Affinity Column;130
14.7;Lab Session 13A. Interim Laboratory Session;131
14.7.1;Laboratory Exercise;131
14.7.1.1;Inoculate Cultures for Protein Purification;131
14.8;Lab Session 13B. Extraction of Recombinant Protein from Escherichia coli and Purification Using a Glutathione Affinity Column;132
14.8.1;Introduction;132
14.8.2;Laboratory Exercises;135
14.8.2.1;Growing Bacterial Suspension Cultures for Fusion Protein Purification;135
14.8.2.2;Harvesting IPTG-Induced Cultures;135
14.8.2.3;Breaking Open Bacterial Cells;135
14.8.2.4;Removing Insoluble Debris from the Crude Homogenate;136
14.8.2.5;Purifying Protein by Affinity Chromatography;136
14.9;Lab Session 13. Discussion Questions;138
14.10;Lab Session 14. Analysis of Purification Fractions;140
14.11;Lab Session 14A. Analysis of Purification Fractions;141
14.11.1;Introduction;141
14.11.2;Laboratory Exercises;142
14.11.2.1;SDS-PAGE of Purified Fusion Protein;142
14.11.2.2;Fluorescence Analysis of Affinity Purification;143
14.12;Lab Session 14B. Replica Plate Positive Clone;147
14.13;Lab Session 14. Discussion Questions;147
15;4 – Analysis of mRNA Levels;148
15.1;Challenges of Working with RNA;149
15.2;References;150
15.3;Lab Session 15. Total RNA Purification;152
15.4;Lab Session 15A. Interim Laboratory Session;153
15.4.1;Laboratory Exercise;153
15.4.1.1;Inoculate Cultures for RNA Purification;153
15.5;Lab Session 15B. Total RNA Purification;154
15.5.1;Introduction;154
15.5.2;Laboratory Exercises;156
15.5.2.1;Purification of Total RNA;156
15.5.2.2;DNase Digestion;158
15.5.2.3;Quantification of RNA;158
15.5.3;References;159
15.6;Lab Session 15. Discussion Questions;159
15.7;Lab Session 16. Analysis of gst::egfp mRNA Levels by RT-qPCR: Part 1;160
15.7.1;Introduction;160
15.7.1.1;Reverse Transcription;161
15.7.1.2;Quantitative PCR;162
15.7.2;Laboratory Exercises;164
15.7.2.1;Reverse Transcription;164
15.7.2.2;Quantitative PCR (qPCR);165
15.7.3;Discussion Questions;167
15.7.4;Reference;167
15.8;Lab Session 17. Analysis of gst::egfp mRNA Levels by RT-qPCR: Part 2;168
15.8.1;Introduction;168
15.8.2;Laboratory Exercise;171
15.8.2.1;Relative Quantification of gst::egfp Levels;171
15.8.3;References;173
15.8.4;Discussion Questions;173
15.9;Lab Session 18. Analysis of gst::egfp mRNA Levels by Semi-Quantitative RT-PCR: Part 1;174
15.9.1;Introduction;174
15.9.2;Laboratory Exercises;175
15.9.2.1;Reverse Transcription (RT);175
15.9.2.2;Semi-Quantitative PCR;175
15.9.3;Discussion Questions;178
15.10;Lab Session 19. Analysis of gst::egfp mRNA Levels by Semi-Quantitative RT-PCR: Part 2;180
15.10.1;Introduction;180
15.10.2;Laboratory Exercises;180
15.10.2.1;Agarose Gel Electrophoresis;180
15.10.2.2;Quantification;181
15.10.3;Discussion Questions;182
15.10.4;Reference;182
16;Appendix 1: Equipment;184
16.1;Shared Equipment;184
17;Appendix 2: Prep List;186
17.1;Notes to Prep Staff;186
17.1.1;Plasmids and E. coli Host Strains;186
17.1.2;Antibiotics;186
17.1.3;Aliquots;187
17.2;Bacterial Waste;187
17.2.1;Autoclaving;187
17.2.2;General Lab Preparation;188
17.2.3;Students;188
17.3;Supplies and Reagents;188
17.3.1;Supplies and Reagents for General Use;188
17.3.2;Recipes for General Use;189
17.4;LAB SESSION 1;190
17.4.1;BSA Serial Dilutions and Nitrocellulose Spot Test;190
17.4.2;Recipes;190
17.5;LAB SESSION 2;191
17.5.1;Purification and Digestion of Plasmid (Vector) DNA;191
17.6;LAB SESSION 3;192
17.6.1;PCR Amplification of egfp from pEGFP-N1; Clean-up and Visualization of Digested pET-41a Vector;192
17.6.2;Recipes;193
17.7;LAB SESSION 4;193
17.7.1;Preparation of Insert DNA (egfp) PCR Product;193
17.8;LAB SESSION 5;193
17.8.1;DNA Ligation and Transformation of Escherichia coli;193
17.9;LAB SESSION 6;194
17.9.1;Interim Lab;194
17.10;LAB SESSION 6;195
17.10.1;Colony Hybridization: Monoclonal Antibody Probe;195
17.10.2;Recipes;195
17.11;LAB SESSION 7;196
17.11.1;Characterization of Recombinant Clones;196
17.11.2;Recipes;197
17.11.2.1;Chloronaphthol Stock Solution;197
17.11.2.2;Peroxide Stain;197
17.12;LAB SESSION 8;197
17.12.1;Interim Lab;197
17.13;LAB SESSION 8;198
17.13.1;Characterization of Recombinant Clones: Part 2;198
17.14;LAB SESSION 9;198
17.14.1;Characterization of Recombinant Clones: Part 3;198
17.15;LAB SESSION 10;199
17.15.1;Computational Analysis of DNA Sequence from a Positive Clone: Part 2;199
17.16;LAB SESSION 11;199
17.16.1;Interim Lab;199
17.17; LAB SESSION 11;199
17.17.1;Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot: Part 1;199
17.17.2;Recipes;200
17.18;LAB SESSION 12;201
17.18.1;Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot: Part 2;201
17.18.2;Recipes;202
17.18.2.1;TBS (Tris-buffered Saline, pH 7.6);202
17.19;LAB SESSION 13;202
17.19.1;Interim Lab;202
17.20; LAB SESSION 13;202
17.20.1;Extraction of Recombinant Protein from Escherichia coli Using a Glutathione Affinity Column;202
17.20.2;Recipes;203
17.21;LAB SESSION 14;203
17.21.1;Analysis of Purification Fractions;203
17.21.2;Recipes;204
17.22;LAB SESSION 15;204
17.22.1;Interim Lab;204
17.23;LAB SESSION 15;204
17.23.1;Total RNA Purification;204
17.23.2;Recipes;205
17.24;LAB SESSION 16;206
17.24.1;Analysis of gst::egfp mRNA Levels by RT-qPCR: Part 1;206
17.25;LAB SESSION 17;206
17.25.1;Analysis of gst::egfp mRNA Levels by RT-qPCR: Part 2;206
17.26;LAB SESSION 18;206
17.26.1;Analysis of gst::egfp mRNA Levels by Semi-Quantitative RT-PCR: Part 1;206
17.27;LAB SESSION 19;207
17.27.1;Analysis of gst::egfp mRNA Levels by Semi-Quantitative RT-PCR: Part 2;207
18;Appendix 3: Preparation of Competent E. coli Cells;208
18.1;Introduction;208
18.2;Protocol;208
18.2.1;Preparation of Chemically Competent Cells by Calcium Chloride Treatment;208
18.2.2;Transformation Control;209
19;Appendix 4: Pre-Lab Questions;212
19.1;LAB SESSION 1;212
19.2;LAB SESSION 2;212
19.3;LAB SESSION 3;213
19.4;LAB SESSION 4;213
19.5;LAB SESSION 5;214
19.6;LAB SESSION 6;214
19.7;LAB SESSION 7;215
19.8;LAB SESSION 8;215
19.9;LAB SESSION 9;216
19.10;LAB SESSION 10;216
19.11;LAB SESSION 11;217
19.12;LAB SESSION 12;218
19.13;LAB SESSION 13;218
19.14;LAB SESSION 14;219
19.15;LAB SESSION 15;219
19.16;LAB SESSION 16;220
19.17;LAB SESSION 17;220
19.18;LAB SESSION 18;221
19.19;LAB SESSION 19;222
20;Index;224