A Practical Approach to Molecular Cloning

Table of Contents

• 1.1. Introduction
• 1.2. Laboratory Safety Rules
• 1.2.1. Proper Handling and Storage of Chemical, Biological, and Radiological Reagents
• 1.2.2. Chemical Hazards and Chemical Safety
• 1.2.2.1. Diverse Hazardous Chemicals and General Features of Hazards Associated With Routinely Used Chemicals in the Molecular Biology Laboratory
• 1.2.2.2. Msds (Material Safety Data Sheets)
• 1.2.2.3. General Safety Precautions in Handling Hazardous Chemicals in the Lab
• 1.2.2.4. ‘Dos and Don’Ts’ of Handling Different Types of Hazardous Chemicals
• 1.2.3. Radiochemical Hazards and Radiation Safety
• 1.2.4. Physical Hazards and Physical Safety
• 1.2.4.1. Ultraviolet Radiation
• 1.2.4.2 High-Voltage Electricity
• 1.2.4.3. Cryogenic Hazards Associated With Procedures Involving Extremely Low Temperatures
• 1.2.5. Biological Hazards and Biological Safety
• 1.2.6. Disposal of Hazardous Chemicals & Biological Materials
• General Tips About the Safety and Personal Protection of the Experimenters
• 1.3. Know Your Laboratory
• 1.3.1. Laboratory Equipment & Reagent Orientation
• 1.3.2. Laboratory Equipment Orientation
• 1.3.3. General Instrumentation Facilities
• 1.3.4. Departmental Equipment Facilities
• 1.3.5. Laboratory Equipment Facilities
• 1.4. Practical Requirements for Molecular Biology Research
• 1.4.1. Mathematical Skills Required for the Molecular Biology Laboratory
• Exponential Numbers
• Addition and Subtraction of Exponential Numbers
• Multiplying and Dividing Exponential Numbers
• Determining Significant Figures
• Generally, a Zero is a Significant Figure If:
• 1.4.2. Experimental Skills
• 1.4.2.1 Cleaning Glassware
• 1.4.2.2 Weigh It Right
• 1.4.2.3 Autoclaving
• 1.4.2.4 Micro Pipetting Practice
• 1.4.2.5 Working With Microcentrifuge Tubes and Labeling Them
• 1.4.2.6 Preparation of Laboratory Reagents
• Preparing Parallel Dilutions or Making “X” Solutions:
• Preparing Serial Dilutions
• Steps in Solution Preparation and Several Important Tips
• 1.4.2.7 Gel Loading
• 1.4.2.8 Working With Enzymes
• 1.5. Calibrating Lab Instruments
• 1.5.1. Calibrating a Ph Meter
• 1.5.2. Calibrating and Using An Electronic Balance
• 1.6. Note on Using Kits
• 1.7. Research Strategies for Molecular Biology
• 1.7.1. Gene Cloning in Outline
• 1.7.2. Pcr in Outline
• 1.7.3. The Choice Between Cloning and Pcr
• Basic Techniques Needed for Cloning and Pcr
• Handling Bacteria (Chapter 2)
• Preparation of Dna (Chapters 3 and 4)
• Separating Dna by Gel Electrophoresis (Chapter 5)
• Purifying Dna Molecules from Electrophoresis Gels (Chapter 5)
• Construction of Recombinant Dna Molecules (Chapter 6)
• Introduction of Recombinant Molecules into Host Cells and Recombinant Selection (Chapter 7)
• Cloning Vectors (Chapter 3)
• Conclusion
• Further Reading

• 2.1. Introduction
• 2.2. Categories of Basic Microbiological Techniques
• 2.3. Aseptic Techniques
• 2.3.1. Sterilization
• Protocol 2.1: Procedure for Running An Autoclave
• Materials
• Equipment
• Procedure
• Important Notes and Tips
• 2.3.1.1.B.Radiation Sterilization
• 2.3.1.1.C. Mechanical Sterilization
• 2.3.1.2. Chemical Method (Disinfection)
• 2.3.2. General Rules to Follow in a Microbiology Laboratory
• 2.4. Microbial Culturing Techniques
• 2.4.1. Microbial Growth Media
• (I) Solid Culture Media
• (Ii) Liquid Culture Media
• Protocol 2.2: Procedure to Prepare Minimal Medium
• Materials
• Equipment
• Procedure
• Storage
• Caution
• 2.4.1.B.1. Luria-Bertani Medium
• Protocol 2.3: Procedure to Prepare Lb Medium
• Materials
• Equipment
• Procedure
• Storage
• Caution
• 2.4.1.B.2. 2Xyt Medium
• Protocol 2.4: Procedure to Prepare 2X Yt Medium
• Materials
• Equipment
• Procedure
• Storage
• Caution
• 2.4.1.B.3. Terrific Broth
• Protocol 5: Procedure to Prepare Tb Medium
• Materials
• Equipment
• Procedure
• Storage
• 2.4.1.B.4. Soc Broth
• Protocol 6: Procedure to Prepare Soc Medium
• Materials
• Equipment
• Procedure
• Preparation of Soc Agar Media
• Storage
• Caution
• 2.4.2. Inoculation
• 2.4.3. Isolation
• 2.4.3.1. Common Isolation Techniques
• Protocol 2.7. Procedure for Streaking Culture of Escherichia Coli on Solid Media to Achieve Single Colonies
• Materials
• Equipment
• Procedure
• Spread Plate Method
• Protocol 2.8. Procedure for Spreading the Culture of Escherichia Coli on Solid Media to Achieve Single Colonies.
• Materials
• Equipment
• Procedure
• Culturing of Escherichia Coli
• 2.4.4.1. Growth on Liquid Media
• Protocol 2.9: Procedure to Grow An Overnight Culture of E. Coli
• 69
• Materials
• Equipment
• Procedure
• 2.4.4.1.1B. Growing Larger Cultures
• Protocol 2.10: Procedure to Grow Large Culture of E. Coli
• Materials
• Equipment
• Procedure
• 2.4.4.2. Growth on Solid Media
• 2.4.4.2A. Tittering and Isolating Bacterial Colonies by Serial Dilutions
• Protocol 2.11: Procedure to Carry Out Serial Dilutions and Plating of An Overnight E. Coli Liquid Culture
• Materials
• Equipment
• 2.4.4.2B. Replica Plating
• Protocol 2.12: Procedure to Carry Out Replica Plating of E. Coli Colonies Grown on Lb Agar
• Materials
• Equipment
• Procedure
• 2.4.5. Monitoring the Growth: Bacteria Enumeration
• 2.4.5A. Enumeration With a Count Slide
• Protocol 2.13: Procedure to Determine Total (Viable and Dead)
• Cell Concentrations of E. Coli Cells Growing in Lb Broth
• Materials
• Equipment
• Procedure
• 2.4.5B. Enumeration of Viable Cells by Growing Bacteria on a Solid Medium
• Protocol 2.14: Procedure to Determine Viable Cell Concentration of E. Coli Cells Growing in Lb Broth
• Materials
• Equipment
• Procedure
• 2.4.5C. Enumeration With a Spectrophotometer
• 2.4.5D. The Bacterial Growth Curve
• Protocol 2.15: Procedure to Determine the Growth Curve of E.
• Coli Cells Growing in Lb Broth
• Materials
• Equipment
• Procedure
• 2.4.6. Preservation of Stock Cultures
• 2.4.6A. Preservation of Short-Term Cultures
• 2.4.6B. Stab and Slant Cultures
• Protocol 2.16: Procedure for the Preparation of Stab Culture of E.
• Coli for Preservation
• Materials
• Equipment
• Procedure
• 2.4.6C. Preservation of Cultures With Glycerol or Dmso
• Protocol 2.17: Procedure for the Preparation of Glycerol Stock of E. Coli for Long-Term Preservation
• Materials
• Equipment
• Procedure
• Conclusion
• Further Reading

• 3.1. Introduction to Plasmid Vectors
• 3.1.1. Plasmids as Cloning Vehicles
• Origin of Replication
• Selectable Marker
• Cloning Site
• 3.1.2. Types of Plasmids
• Relaxed Plasmids
• Stringent Plasmids
• 3.2. Isolation of Plasmid Dna
• 3.2.1. Isolation and Purification of Plasmid Dna by Alkaline Lysis

Author

• Satarupa Das
• Biswadip Das