Molecular Biology: A Project Approach
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Table of contents
- CONTENTSvii
- METHODS LOCATORxiii
- SUGGESTED SCHEDULE OF LABORATORY PROTOCOLSxv
- PREFACExvii
- ACKNOWLEDGMENTSxix
- NOTE TO USERSxxi
- CHAPTER 1. TRANSPOSON MUTAGENESIS OF Escherichia coli1
- Introduction to Transposons1
- Advantages of Transposon Mutagenesis6
- Eukaryotic Transposable Elements8
- Transposons and Gene Fusions9
- Preparing for Laboratory Exercises9
- Tn5 Mutagenesis of Escherichia coil and Analysis of Auxotrophs: Overview15
- Protocol 1.1: Phage λ Titer19
- Protocol 1.2: Making a Phage Stock—Growing λ-Tn5'21
- Protocol 1.3: Transposon Mutagenesis Using λ::TnphoA'-225
- Introduction to Auxotrophs27
- Protocol 1.4: Isolation of Auxotrophs–Replica Plating, Toothpicking, or Screening on 2 EM Plates27
- Protocol 1.5: Identification of Auxotrophs on Pool Plates31
- Protocol 1.6: Analysis of Auxotrophs Using a Literature Search33
- References35
- Suggested Reading36
- CHAPTER 2. RECOMBINANT DNA CLONING45
- Introduction to Recombinant DNA Technology45
- Cloning Vectors49
- Restriction Endonucleases63
- The Use of Restriction Endonucleases: Practical Matters71
- Ligase77
- Gel Electrophoresis78
- Ethidium Bromide Staining of DNA in Gels85
- Ethidium Bromide Safety86
- Sensitivity of Detection with Ethidium Bromide87
- Other DNA Stains87
- Transformation88
- Recombinant DNA Cloning: Overview92
- Recombinant DNA: P1 Level of Physical Containment„Laboratory Practices97
- Protocol 2. la (Optional): Restriction Digestion of DNA Samples and Gel Electrophoresis of DNA Sampl97
- Protocol 2.1 b (Optional): Restriction Enzyme Digestion of DNA to Be Cloned98
- Protocol 2.2 (Optional): Gel Electrophoresis99
- Protocol 2.3: Large-Scale Plasmid Isolation Using Alkaline Lysis102
- Protocol 2.4: Recombinant DNA Cloning110
- Protocol 2.5a: Competent Escherichia coli Cells116
- Protocol 2.5b: Preparing Fresh Competent Escherichia coli Cells for Transformation117
- Protocol 2.5c: A Rapid Colony Transformation Procedure119
- Protocol 2.6a: Boiling Mini-Prep Isolation of Plasmid DNA120
- Protocol 2.6b: Alkaline Mini-Prep Procedure for Isolating Plasmid DNA121
- References123
- Suggested Reading131
- CHAPTER 3. SOUTHERN BLOT ANALYSIS135
- Southern Blot Introduction135
- Using Southern Blot Analysis to Map Restriction Endonuclease Sites136
- Nonradioactive Labeling of Nucleic Acids136
- Autoradiography: Overview143
- Isolation of Nucleic Acid Fragments from Gels145
- Labeling Methods147
- Hybridization to Membranes153
- The Attachment of Nucleic Acids to a Membrane156
- Protocol 3.1 a: Southern Blot157
- Protocol 3.1 b: Bidirectional Blotting: A Sandwich Blot161
- Protocol 3.1 c: Alkaline Blotting161
- Protocol 3.1 d: Colony Hybridization162
- Protocol 3.2: Isolation of DNA Fragments by Electroelution164
- Protocol 3.3a: Labeling Probe with Biotin Using Nick Translation169
- Protocol 3.3b: Oligo Labeling of a Probe171
- Protocol 3.3c: Protobiotin Labeling of a Probe173
- Protocol 3.4a: Hybridization and Detection of Labeled Probe„A Biotin-Labeled Nonradioactive Probe174
- Protocol 3.4b: Hybridization and Detection of Labeled Probe„A Biotin-Labeled Nonradioactive Probe178
- Protocol 3.5: Standard Southern Blot Hybridization with 32p-Labeled Probe187
- References189
- Suggested Reading192
- CHAPTER 4. PLANT GENOMIC SOUTHERN BLOTTING WITH PROBES FOR LOW- AND HIGH-COPY-NUMBER GENES193
- Overview of Experiment193
- Protocol 4.1: Plant DNA Extraction Mini-Prep Procedure195
- "Reconstructions" for Gels196
- Protocol 4.2: Steps of a Genomic Southern Blot199
- References200
- Suggested Reading200
- CHAPTER 5. RNA PURIFICATION AND NORTHERN BLOT ANALYSIS202
- RNA Introduction: Overview of Experiment202
- Protocol 5.1: RNA Extraction from Plant Leaves203
- Protocol 5.2: Separating Poly(A) + RNA from Total Cellular RNA206
- Protocol 5.3: RNA Gel: A Denaturing Formaldehyde Gel207
- Protocol 5.4: A Northern Blot208
- Protocol 5.5: Standard Northern Blot Hybridization Conditions for 32p-Labeled Probe210
- Protocol 5.6: Nonradioactive Biotin-Labeled Probes for Northern Blots213
- References213
- Suggested Reading213
- CHAPTER 6. POLYMERASE CHAIN REACTION215
- Background215
- Protocol 6.1: PCR Experiment219
- References223
- Suggested Reading224
- APPENDIX 1: Templates for Streaking Colonies229
- APPENDIX 2: Storing Bacterial Strains: Making Permanents231
- APPENDIX 3: Reporter Genes235
- APPENDIX 4: Antibiotic Information239
- APPENDIX 5: X-gal and IPTG247
- APPENDIX 6: More Information on Molecular Biology Protocols249
- APPENDIX 7: Sources of Strains251
- APPENDIX 8: List of Suppliers253
- APPENDIX 9: Additional Information257
- APPENDIX 10: Molecular Weight Standards259
- GLOSSARY261
- INDEX275
This course manual instructs students in recombinant DNA techniques and other essential molecular biology techniques in the context of projects. The project approach inspires and captivates students; it involves them in the scientific experience, providing continuity to laboratory bench time and an understanding of the principles underlying the techniques presented.
Molecular Biology is a must for any department, operating under budgetary constraints that offers or plans to offer a course in molecular cloning.
Key Features
* Includes a glossary of over 200 terms important for understanding molecular biology
* Uses an inexpensive source of eukaryotic cells - great for schools on a budget
* Includes Methods Locator that provides instant access to the latest methods
* Contain clearly written, easy-to-follow, student-tested instructions:
* Sterile techniques
* Phage titration
* Gel electrophoresis of DNA
* Restriction enzyme digestion
* Plasmid isolation
* Transformation of E. Coli
* Recombinant DNA cloning
* Nick translation labeling
* Nonradioactive primer labelling
* Nonradioactive DNA detection
* Southern blotting
* Colony hybridization
* Purification of plant DNA
* RNA purification
* Northern blotting
* Purification of poly A+ RNA
* Polymerase chain reaction (PCR)
Molecular Biology is a must for any department, operating under budgetary constraints that offers or plans to offer a course in molecular cloning.
Key Features
* Includes a glossary of over 200 terms important for understanding molecular biology
* Uses an inexpensive source of eukaryotic cells - great for schools on a budget
* Includes Methods Locator that provides instant access to the latest methods
* Contain clearly written, easy-to-follow, student-tested instructions:
* Sterile techniques
* Phage titration
* Gel electrophoresis of DNA
* Restriction enzyme digestion
* Plasmid isolation
* Transformation of E. Coli
* Recombinant DNA cloning
* Nick translation labeling
* Nonradioactive primer labelling
* Nonradioactive DNA detection
* Southern blotting
* Colony hybridization
* Purification of plant DNA
* RNA purification
* Northern blotting
* Purification of poly A+ RNA
* Polymerase chain reaction (PCR)