Molecular biology techniques: a classroom laboratory manual
Gespeichert in:
| Hauptverfasser: | , , |
|---|---|
| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
Amsterdam [u.a.]
Elsevier, Acad. Press
2012
|
| Ausgabe: | 3. ed. |
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | XXVI, 200 S. Ill., graph. Darst. |
| ISBN: | 9780123855442 |
Internformat
MARC
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| 245 | 1 | 0 | |a Molecular biology techniques |b a classroom laboratory manual |c Susan Carson ; Heather Miller ; D. Scott Witherow |
| 250 | |a 3. ed. | ||
| 264 | 1 | |a Amsterdam [u.a.] |b Elsevier, Acad. Press |c 2012 | |
| 300 | |a XXVI, 200 S. |b Ill., graph. Darst. | ||
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Datensatz im Suchindex
| _version_ | 1804148449437810688 |
|---|---|
| adam_text | Titel: Molecular biology techniques
Autor: Carson, Sue
Jahr: 2012
Contents
Preface xi
About the Authors xiii
Acknowledgements xv
Note to Instructors xvii
Instrumentation xix
Nomenclature xxi
Introduction xxiii
Conceptual Outline for Experiments xxiii
Experimental Procedures xxiii
Laboratory Safety xxv
General Operating Procedures xxvi
PART 1 - Manipulation of DNA 1
Reference 2
Lab Session 1 Getting Oriented: Practicing with Micropipettes 3
Station Checklist 3
Micropipetting 4
Micropipetting Self-Test 6
Laboratory Exercise: BSA Serial Dilutions and Nitrocellulose Spot Test 7
Preparing BSA Dilutions 7
Performing a Nitrocellulose Spot Test 7
Discussion Questions 9
Lab Session 2 Purification and Digestion of Plasmid (Vector) DNA 11
Introduction to Plasmid Purification 11
Alkaline Lysis 11
Silica Adsorption 12
DNA Quantification 12
Introduction to Expression Vectors 12
Principles of Gene Expression 13
Expression Vectors 13
Orientation and Reading Frame 14
Orientation 14
Reading Frame 15
Laboratory Exercises 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 21
Introduction 21
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
Laboratory Exercises 25
PCR Amplification of egfp from the pEGFP-N 1 Plasmid 25
PCR Protocol 25
Clean-up of Digested pET-41a 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 35
Introduction 35
Ligation 35
Transformation 36
Laboratory Exercises 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
Lab Session 6A Interim Laboratory Session 44
Introduction 44
Laboratory Exercises 44
Counting Transformants and Replica Plating 44
Replica Plating 44
Lab Session 6B Colony Hybridization: Monoclonal Antibody Probe 46
Introduction 46
Laboratory Exercises 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 52
Introduction 52
Laboratory Exercise 52
Colony Hybridization with an a-EGFP Monoclonal Antibody Probe: Part 2 52
Lab Session 7B PCR Screening 54
Introduction 54
Laboratory Exercise 54
Polymerase Chain Reaction Screen for Recombinant Clones 54
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 60
Laboratory Exercise 60
Inoculate Cultures for Minipreps 60
Lab Session 8B Analysis of PCR Screen Results 61
Introduction 61
Laboratory Exercise 61
Gel Electrophoresis and Analysis of PCR Samples from Last Week 61
Lab Session 8C Isolation of Miniprep DNA from Potential Transformants 62
Introduction 62
Laboratory Exercise 63
Isolation of Miniprep DNA from Potential Transformants 63
Plasmid DNA Purification Using the QIAprep Spin Miniprep Kit
and a Microcentrifuge 63
Lab Session 8D Visualization of Green Fluorescent Protein: Part 1 65
Introduction 65
Laboratory Exercise 65
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) 68
Introduction 68
Laboratory Exercise 68
Restriction Enzyme Analysis of Miniprep DNA 68
Lab Session 9B Visualization of Green Fluorescent Protein: Part 2 70
Introduction 70
Laboratory Exercise 70
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 1 72
Introduction 72
Laboratory Exercise 74
References 76
Lab Session 9 Discussion Questions 76
Lab Session 10 Computational Analysis of DNA Sequence from a Positive Clone: Part 2 77
Introduction 77
Laboratory Exercise 81
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 90
Laboratory Exercise 90
Inoculate Cultures for SDS-PAGE 90
Lab Session 11B Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot 91
Introduction 91
Laboratory Exercise 93
SDS-PAGE and Western Blot: Part 1 93
Reference 97
Lab Session 11 Discussion Questions 97
Lab Session 12 Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot: Part 2 99
Introduction 99
Laboratory Exercises 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 104
Laboratory Exercise 104
Inoculate Cultures for Protein Purification 104
Lab Session 13B Extraction of Recombinant Protein from Escherichia coli and Purification Using a
Glutathione Affinity Column 105
Introduction 105
Laboratory Exercises 108
Growing Bacterial Suspension Cultures for Fusion Protein Purification 108
Harvesting IPTG-Induced Cultures 108
Breaking Open Bacterial Cells 108
Removing Insoluble Debris from the Crude Homogenate 108
Purifying Protein by Affinity Chromatography 109
Lab Session 13 Discussion Questions 109
Lab Session 14 Analysis of Purification Fractions 111
Lab Session 14A Analysis of Purification Fractions 113
Introduction 114
Laboratory Exercises 114
SDS-PAGE of Purified Fusion Protein 115
Fluorescence Analysis of Affinity Purification 115
Lab Session 14B Replica Plate Positive Clone 116
Lab Session 14 Discussion Questions 120
PART 4 - Analysis of mRNA Levels 120
Challenges of Working with RNA 121
References 122
Lab Session 15 Total RNA Purification 123
Lab Session 15A Interim Laboratory Session 125
Laboratory Exercise 126
Inoculate Cultures for RNA Purification 126
Lab Session 15B Total RNA Purification 127
Introduction 127
Laboratory Exercises 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 1 133
Introduction 133
Reverse Transcription 134
Quantitative PCR 135
Laboratory Exercises 137
Reverse Transcription 137
Quantitative PCR (qPCR) 138
Discussion Questions 140
Reference 140
Lab Session 17 Analysis oi gstr.egfp mRNA Levels by RT-qPCR: Part 2 141
Introduction 141
Laboratory Exercise 144
Relative Quantification of gstr.egfp Levels 144
References 146
Discussion Questions 146
Lab Session 18 Analysis of gstr::egfp mRNA Levels by Semi-Quantitative RT-PCR: Part 1 147
Introduction 147
Laboratory Exercises 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-PCR: Part 2 153
Introduction 153
Laboratory Exercises 153
Agarose Gel Electrophoresis 153
Quantification 154
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
|
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| isbn | 9780123855442 |
| language | English |
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| physical | XXVI, 200 S. Ill., graph. Darst. |
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| spelling | Carson, Sue Verfasser (DE-588)1018606211 aut Molecular biology techniques a classroom laboratory manual Susan Carson ; Heather Miller ; D. Scott Witherow 3. ed. Amsterdam [u.a.] Elsevier, Acad. Press 2012 XXVI, 200 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Methode (DE-588)4038971-6 gnd rswk-swf Molekularbiologie (DE-588)4039983-7 gnd rswk-swf Molekularbiologie (DE-588)4039983-7 s Methode (DE-588)4038971-6 s DE-604 Miller, Heather B. Verfasser (DE-588)1018607323 aut Witherow, D. Scott Verfasser (DE-588)1018608419 aut HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=024458541&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Carson, Sue Miller, Heather B. Witherow, D. Scott Molecular biology techniques a classroom laboratory manual Methode (DE-588)4038971-6 gnd Molekularbiologie (DE-588)4039983-7 gnd |
| subject_GND | (DE-588)4038971-6 (DE-588)4039983-7 |
| title | Molecular biology techniques a classroom laboratory manual |
| title_auth | Molecular biology techniques a classroom laboratory manual |
| title_exact_search | Molecular biology techniques a classroom laboratory manual |
| title_full | Molecular biology techniques a classroom laboratory manual Susan Carson ; Heather Miller ; D. Scott Witherow |
| title_fullStr | Molecular biology techniques a classroom laboratory manual Susan Carson ; Heather Miller ; D. Scott Witherow |
| title_full_unstemmed | Molecular biology techniques a classroom laboratory manual Susan Carson ; Heather Miller ; D. Scott Witherow |
| title_short | Molecular biology techniques |
| title_sort | molecular biology techniques a classroom laboratory manual |
| title_sub | a classroom laboratory manual |
| topic | Methode (DE-588)4038971-6 gnd Molekularbiologie (DE-588)4039983-7 gnd |
| topic_facet | Methode Molekularbiologie |
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