Manipulation and expression of recombinant DNA : a laboratory manual

Preface xi Acknowledgments xiii Note to Instructors xv Instrumentation xvi Nomenclature xvi INTRODUCTION Conceptual Outline for Experiments 1 I. EXPERIMENTAL PROCEDURES 1 II. LABORATORY SAFETY 2 III. GENERAL OPERATING PROCEDURES 4 IV. EMERGENCY CONTACT INFORMATION 4 PART I Manipulation of DNA LAB SESSION 1 Getting Oriented; Practicing with Micropipettes 7 I. STATION CHECKLIST 7 II. MICROPIPETTING 9 III. LABORATORY EXERCISE 11 A. Preparing BSA Dilutions 11 B. Performing a Nitrocellulose Spot Test 12 LAB SESSION 2 Large-Scale Purification of Plasmid DNA 13 I. INTRODUCTION 13 II. LABORATORY EXERCISES 14 A. Alkaline Lysis and Anion Exchange Chromatograhy 14 B. DNA Quantification 17 LAB SESSION 3 Preparation of Expression Vector DNA (pET-41a(+), a GST Fusion Protein Vector) 19 I. INTRODUCTION 19 II. LABORATORY EXERCISES 23 A. Restriction Digestion of Vector (pET41a) Restriction Enzyme Digestions 23 B. Agarose Gel Electrophoresis 24 C. Cleaning DNA Using a Spin Column 26 LAB SESSION 4 Preparation of Insert DNA (egfp) 29 I. INTRODUCTION 29 II. LABORATORY EXERCISES 30 A. Restriction Digestion of pEGFP-N1 30 B. Isolation of egfp DNA from Agarose 30 LAB SESSION 5 Preparation of Transformation-Competent Cells and Control Transformation 35 I. INTRODUCTION 35 II. LABORATORY EXERCISES 35 A. Preparation of Chemically Competent Cells by Calcium Chloride Treatment 35 B. Transformation Control 36 LAB SESSION 6 DNA Ligation and Transformation of Escherichia coli 39 I. INTRODUCTION 39 II. LABORATORY EXERCISES 41 A. Ligations and Ligation Controls 41 B. Divalent Cation-Mediated Transformation 42 C. Electrophoresis of Ligation Reactions 43 vi Contents PART II Screening Transformants LAB SESSION 7 Colony Hybridizations 47 LAB SESSION 7A Interim Laboratory Session 49 I. INTRODUCTION 49 II. LABORATORY EXERCISES 49 A. Counting Transformants 49 B. Replica Plating 50 LAB SESSION 7B Colony Hybridization: DNA Probe 53 I. INTRODUCTION 53 II. LABORATORY EXERCISES 57 A. Colony Hybridization with an egfp DNA Probe: Part 1 57 B. Labeling of DNA Probe by PCR Using Digoxigenin-11-dUTP 57 LAB SESSION 7C Colony Hybridization: Monoclonal Antibody Probe 59 I. INTRODUCTION 59 II. LABORATORY EXERCISE 60 A. Colony Hybridization with an a-GFP Monoclonal Antibody Probe: Part 1 60 LAB SESSION 8 Completion of Colony Hybridization with DNA Probe 63 I. INTRODUCTION 63 II. LABORATORY EXERCISE 63 A. Colony Hybridization with an egfp DNA Probe: Part 2 63 LAB SESSION 9 Characterization of Recombinant Clones 65 LAB SESSION 9A Completion of Colony Hybridization with mAB Probe 67 I. INTRODUCTION 67 II. LABORATORY EXERCISE 67 A. Colony Hybridization with an α-GFP monoclonal Antibody Probe: Part 2 67 Contents vii LAB SESSION 9B PCR Screening 69 I. INTRODUCTION 69 II. LABORATORY EXERCISE 71 A. Polymerase Chain Reaction Screen for Recombinant Clones: Part 1 71 LAB SESSION 9C Visualization of Green Fluorescent Protein: Part 1 73 I. INTRODUCTION 73 II. LABORATORY EXERCISE 73 A. Green Fluorescence Assay and Preparation of a Fresh Master Plate 73 LAB SESSION 10 Further Characterization of Recombinant Clones 75 LAB SESSION 10A: Interim Laboratory Session 77 I. LABORATORY EXERCISE 77 A. Inoculate Cultures for Minipreps 77 LAB SESSION 10B Analysis of PCR Screen Results 79 I. INTRODUCTION 79 II. LABORATORY EXERCISE 79 A. Gel Electrophoresis and Analysis of PCR Samples 79 LAB SESSION 10C Isolation and Characterization of Miniprep DNA from Potential Transformants (Restriction Analysis of Putative Transformants) 81 I. INTRODUCTION 81 II. LABORATORY EXERCISES 82 A. Alkaline Lysis and Ethanol Precipitation of Miniprep DNA 82 B. Restriction Enzyme Analysis of Miniprep DNA 83 LAB SESSION 10D Visualization of Green Fluorescent Protein: Part 2 85 I. INTRODUCTION 85 II. LABORATORY EXERCISE 85 A. Visualization of Clones Expressing the Enhanced Green Fluorescent Protein on IPTG Plates 85 viii Contents PA R T I I I Expression, Detection, and Purification of Recombinant Proteins from Bacteria LAB SESSION 11 Expression of Fusion Protein from Positive Clones and Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunological Analysis (Western Blot): Part 1 91 LAB SESSION 11A Interim Laboratory Session 93 I. LABORATORY EXERCISE 93 A. Inoculate Cultures for SDS-PAGE 93 LAB SESSION 11B Expression of Fusion Protein from Positive Clones and Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunological Analysis (Western Blot): Part 1 95 I. INTRODUCTION 95 II. LABORATORY EXERCISE 96 A. SDS-Polyacrylamide Gel Electrophoresis and Western Blot: Part 1 96 LAB SESSION 12 Expression of Fusion Protein from Positive Clones and Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunological Analysis (Western Blot): Part 2 101 I. INTRODUCTION 101 II. LABORATORY EXERCISES 102 A. SDS-Polyacrylamide Gel Electrophoresis and Western Blot: Part 2 102 B. Replica Plate Positive Clone 103 LAB SESSION 13 Extraction of Recombinant Protein from Escherichia coli Using a Glutathione Affinity Column 105 LAB SESSION 13A Interim Laboratory Session 107 I. LABORATORY EXERCISE 107 A. Inoculate Cultures for Protein Purification 107 Contents ix LAB SESSION 13B Extraction of Recombinant Protein from Escherichia coli Using a Glutathione Affinity Column 109 I. INTRODUCTION 109 II. LABORATORY EXERCISES 112 A. Growing Bacterial Suspension Cultures for Fusion Protein Purification 112 B. Harvesting IPTG-Induced Cultures 112 C. Breaking Open Bacterial Cells 112 D. Removing Insoluble Debris from the Crude Homogenate 113 E. Purifying Protein by Affinity Chromatography 113 LAB SESSION 14 Analysis of Purification Fractions 115 I. INTRODUCTION 115 II. LABORATORY EXERCISES 117 A. SDS-PAGE of purified Fusion Protein 117 B. Bradford Protein Concentration Determination Assay of Purification Fractions 118 APPENDIXES 1. EQUIPMENT 121 2. PREP LIST 123 3. MAKING SENSE OF ORIENTATION 143 INDEX 147