Molecular Biology Techniques: A Classroom Laboratory Manual THIRD EDITION

Transcription

1 Molecular Biology Techniques: A Classroom Laboratory Manual THIRD EDITION Susan Carson Heather B. Miller D.Scott Witherow ELSEVIER AMSTERDAM BOSTON HEIDELBERG LONDON NEW YORK OXFORD PARIS SAN DIEGO SAN FRANCISCO SINGAPORE SYDNEY TOKYO Academic Press is an imprint of Elsevier

2 Preface About the Authors Acknowledgements Note to Instructors Instrumentation Nomenclature Conceptual Outline for Experiments Experimental Procedures Laboratory Safety General Operating Procedures XI xiii xv xvii xix xxi xxiii xxiii xxiii xxv xxvi PART 1 - Manipulation of I Lab Session 1 Lab Session 2 Reference Getting Oriented: Practicing with Micropipettes Station Checklist Micropipetting Micropipetting Self-Test : BSA Serial Dilutions and Nitrocellulose Spot Test Preparing BSA Dilutions Performing a Nitrocellulose Spot Test Discussion Questions Purification and Digestion of Plasmid (Vector) DNA to Plasmid Purification Alkaline Lysis Silica Adsorption DNA Quantification to Expression Vectors Principles of Gene Expression Expression Vectors

3 Orientation and Reading Frame 14 Orientation 14 Reading Frame 15 s 16 Alkaline Lysis and Silica Adsorption Protocol 16 DNA Quantification 18 Restriction Digestion of Expression Vector DNA pet-41a, a GST Fusion Protein Vector 19 Discussion Questions 20 Reference 20 Lab Session 3 PCR Amplification of egfp and Completion of Vector Preparation What is the Polymerase Chain Reaction (PCR)? 21 Why Clone by PCR? 23 TA Cloning 23 PCR Cloning by Incorporation of Restriction Sites 23 Cloning Synthetic Genes 24 s 25 PCR Amplification of egfp from the pegfp-nl Plasmid 25 PCR Protocol 25 Clean-up of Digested pet-4la Vector 25 Agarose Gel Electrophoresis 27 Discussion Questions 29 References 29 Lab Session 4 Preparation of Insert DNA {egfp) PCR Product 31 Check PCR Reactions on an Agarose Gel 31 Spin Column Cleanup of PCR Product 31 Quantification of egfp PCR Product 31 Restriction Digestion of egfp PCR Product 32 Removing Enzymes and Cleaning Digested DNA Using a Spin Column 32 Discussion Questions 33 Lab Session 5 DNA Ligation and Transformation of Escherichia coli Ligation 35 Transformation 36 s 37 Ligations and Ligation Controls 37 Divalent Cation-Mediated Transformation 39 Electrophoresis of Ligation Reactions 39 Discussion Questions 40 Reference 40 PART 2 - Screening Transformants 41 Lab Session 6 Colony Hybridization 43

4 Lab Session 6A Interim Laboratory Session s 44 Counting Transformants and Replica Plating 44 Replica Plating 44 Lab Session 6B Colony Hybridization: Monoclonal Antibody Probe s 47 Colony Hybridization with an a-egfp Monoclonal Antibody Probe: Part 1 47 Lab Session 6 Discussion Questions 49 Lab Session 7 Characterization of Recombinant Clones: Part 1 51 Lab Session 7A Completion of Colony Hybridization with a Monoclonal Antibody Probe Lab Session 7B PCR Screening Colony Hybridization with an a-egfp Monoclonal Antibody Probe: Part 2 Polymerase Chain Reaction Screen for Recombinant Clones Cffl Lab Session 7C Prepare Fresh Replica Plate 57 Lab Session 7 Discussion Questions 57 Lab Session 8 Characterization of Recombinant Clones: Part 2 59 Lab Session 8A Interim Laboratory Session Inoculate Cultures for Minipreps 60 Lab Session 8B Analysis of PCR Screen Results Gel Electrophoresis and Analysis of PCR Samples from Last Week 61 Lab Session 8C Isolation of Miniprep DNA from Potential Transformants Isolation of Miniprep DNA from Potential Transformants 63 Plasmid DNA Purification Using the QIAprep Spin Miniprep Kit and a Microcentrifuge 63

5 Lab Session 8D Visualization of Green Fluorescent Protein: Part Green Fluorescence Assay and Preparation of a Fresh Master Plate 65 Lab Session 8 Discussion Questions 66 Lab Session 9 Characterization of Recombinant Clones: Part 3 67 Lab Session 9A Characterization of Miniprep DNA from Potential Transformants (Restriction Enzyme Analysis of Putative Transformants) Restriction Enzyme Analysis of Miniprep DNA 68 Lab Session 9B Visualization of Green Fluorescent Protein: Part Visualization of Clones Expressing the Enhanced Green Fluorescent Protein on IPTG Plates 70 Lab Session 9C Computational Analysis of DNA Sequence from a Positive Clone: Part References 76 Lab Session 9 Discussion Questions 76 Lab Session 10 Computational Analysis of DNA Sequence from a Positive Clone: Part Discussion Questions 85 Reference 85 PART 3 - Expression, Detection and Purification of Recombinant Proteins from Bacteria 87 Lab Session 11 Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot: Part 1 89 Lab Session 11A Interim Laboratory Session Inoculate Cultures for SDS-PAGE 90 Lab Session 11B Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot SDS-PAGE and Western Blot: Part 1 93 Reference 97 Lab Session 11 Discussion Questions 97

6 Lab Session 12 Expression of Fusion Protein from Positive Clones, SDS- PAGE and Western Blot: Part s 99 SDS-PAGE and Western Blot: Part 2 99 Replica Plate Positive Clone 101 Discussion Questions 101 Lab Session 13 Extraction of Recombinant Protein from Escherichia coli Using a Glutathione Affinity Column 103 Lab Session 13A Interim Laboratory Session Inoculate Cultures for Protein Purification Lab Session 13B Extraction of Recombinant Protein from Escherichia coli and Purification Using a Glutathione Affinity Column s Growing Bacterial Suspension Cultures for Fusion Protein Purification Harvesting IPTG-Induced Cultures Breaking Open Bacterial Cells Removing Insoluble Debris from the Crude Homogenate Purifying Protein by Affinity Chromatography Lab Session 13 Discussion Questions Lab Session 14 Analysis of Purification Fractions Lab Session 14A Analysis of Purification Fractions s SDS-PAGE of Purified Fusion Protein Fluorescence Analysis of Affinity Purification Lab Session 14B Replica Plate Positive Clone Lab Session 14 Discussion Questions PART 4 - Analysis of mrna Levels Challenges of Working with RNA References Lab Session 15 Total RNA Purification Lab Session 15A Interim Laboratory Session Inoculate Cultures for RNA Purification

7 Lab Session 15B Total RNA Purification s 129 Purification of Total RNA 129 DNase Digestion 131 Quantification of RNA 131 References 132 Lab Session 15 Discussion Questions 132 Lab Session 16 Analysis of gstr.egfp mrna Levels by RT-qPCR: Part Reverse Transcription 134 Quantitative PCR 135 s 137 Reverse Transcription 137 Quantitative PCR (qpcr) 138 Discussion Questions 140 Reference 140 Lab Session 17 Analysis of gstr.egfp mrna Levels by RT-qPCR: Part Relative Quantification of gstr.egfp Levels 144 References 146 Discussion Questions 146 Lab Session 18 Analysis of gstregfp mrna Levels by Semi-Quantitative RT-PCR: Part s 148 Reverse Transcription (RT) 148 Semi-Quantitative PCR 148 Discussion Questions 151 Lab Session 19 Analysis of gstr.egfp mrna Levels by Semi-Quantitative RT-1 'CR: Part s 153 Agarose Gel Electrophoresis 153 Quantification 1 Discussion Questions 155 Reference 155 Appendix 1: Equipment 157 Appendix 2: Prep List 159 Appendix 3: Preparation of Competent E. coli Cells 181 Appendix 4: Pre-Lab Questions 185 Index 197