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Table of Contents
Intro
Advanced Methods in Molecular Biology and Biotechnology: A Practical Lab Manual
Copyright
Dedication
Contents
Preface
Foreword
Acknowledgment
Introduction
About the author
Disclaimer
Chapter 1 Introduction to molecular biology techniques
Safety measures
Preparation of the solutions
Working with the nucleic acids
Storage
Purification
Quantitation
Concentration
Restriction enzymes
Sterile techniques
References
Chapter 2 Agarose gel electrophoresis
Definition
Rationale
Before you begin
Key resources table
Materials and equipments
Materials and equipments required
Reagents required
Step-by-step method details
Timing: 1-1.5 h (depending on the size of the sample)
Expected outcomes
Quantification and statistical analysis
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Alternative methods/procedures
References
Chapter 3 SDS-polyacrylamide gel electrophoresis
Definition
Rationale
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Timing: 3-4 h (depends on the plate size)
Expected outcomes
Quantification and statistical analysis
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Alternative methods/procedures
References
Chapter 4 Immunoelectrophoresis
Definition
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected outcomes
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
References.
Chapter 5 Plasmid DNA isolation using alkaline lysis method from E. coli DH5 α cells
Definition and rationale
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected outcomes
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Alternative methods/procedures
References
Chapter 6 Restriction digestion of plasmid DNA
Definition and rationale
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected outcomes
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
References
Chapter 7 Genomic DNA extraction from the plant leaves using the CTAB method
Definition
Rationale
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected outcomes
Quantification and statistical analysis
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Alternative methods/procedures
References
Chapter 8 Genomic DNA extraction from silkworm using modified phenol:chloroform method (Suzuki et al., 1972)
Definition and rationale
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected outcomes
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Alternative methods/procedures
References
Chapter 9 Genomic DNA extraction from Fungal Mycelium using the modified Cenis, 1992 method
Definition and rationale
Before you begin
Key resources table
Materials and equipment.
Step-by-step method details
Expected outcomes
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Alternative methods/procedures
References
Chapter 10 Bacterial transformation using CaCl 2
Definition
Rationale
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected outcomes
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Alternative methods/procedures
References
Chapter 11 Transformation of E. coli by electroporation
Definition
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected outcomes
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Alternative methods/procedures
Further reading
Chapter 12 Transfection of cells using lipofectamine
Definition
Key resources table
Materials and equipments
Step-by-step method details
Advantages
Optimization and troubleshooting
Problem
Potential solution
References
Chapter 13 Total RNA isolation
Definition and rationale
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected outcomes
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Alternative methods/procedures
References
Chapter 14 RNA isolation from plant tissues with high phenolic compounds and polysaccharides
Definition and rationale
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected outcomes.
Quantification and statistical analysis
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Further reading
Chapter 15 Total protein extraction with TCA-acetone and 2D gel electrophoresis
Definition
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected results
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Further reading
Chapter 16 Dansyl-Edman method of peptide sequencing
Definition
Key resources table
Materials and equipment
Step-by-step method details
Advantages
Limitations
Further reading
Chapter 17 ISSR analysis
Definition
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected outcomes
Advantages
Limitations
Safety considerations and standards
Alternative methods/procedures
Further reading
Chapter 18 Gene cloning/recombinant DNA technology
Definition
Rationale
Materials and equipments
Attributes of an ideal vector
Attributes of a good host cell
Step-by-step method details
Plant material
Microbial cultures and plasmids
DEPC treatment
RNA extraction and qRT-PCR
DNase treatment of RNA samples
Quantitative and qualitative analysis of RNA
cDNA synthesis
Primer designing
Validation of cDNA and primers using PCR
PCR reaction mixture
PCR cyclic conditions
Gel electrophoresis
cDNA quantification
Confirmation of cDNA synthesis
Gene cloning
PCR amplification of full-length genes (CBF-1)
Restriction digestion of the PCR fragments and vector
Expression vector
Ligation
Transformation
Confirmation of cloning by restriction digestion.
Heterologous expression
Cell culture experiments
Expected outcomes
Advantages
Limitations
References
Chapter 19 Polymerase chain reaction (PCR)
Definition
Rationale
Types of PCR
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected outcomes
Advantages
Limitations
Applications of PCR
Applications of PCR in agricultural sciences
Optimization and troubleshooting
Problem
Potential solution
Alternative methods/procedures
Further reading
Chapter 20 Blotting/hybridization and its types
Definition
Types of blotting
Step-by-step method details
Applications
Step-by-step method details
Applications
Step-by-step method details
Applications
Step-by-step method details
Applications
Further reading
Chapter 21 Dot blot analysis
Definition and rationale
Before you begin
Materials and equipment
Step-by-step method details
Expected outcomes
Advantages
Limitations
Further reading
Chapter 22 ELISA (enzyme-linked immunosorbent assay)
Definition
Rationale
Types of ELISA
Materials and equipment
Step-by-step method details
Quantification and statistical analysis
Advantages
Limitations
Further reading
Chapter 23 Study of principle of centrifugation
Definition
Types of centrifuge
Before you begin
Materials and equipment
Step-by-step method details
Expected outcomes
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Alternative methods/procedures
Further reading
Chapter 24 Study of principle of chromatography
Definition
Different types of chromatographic techniques
Expected outcomes
Advantages
Limitations
Further reading.
Chapter 25 Thin layer chromatography (TLC).
Advanced Methods in Molecular Biology and Biotechnology: A Practical Lab Manual
Copyright
Dedication
Contents
Preface
Foreword
Acknowledgment
Introduction
About the author
Disclaimer
Chapter 1 Introduction to molecular biology techniques
Safety measures
Preparation of the solutions
Working with the nucleic acids
Storage
Purification
Quantitation
Concentration
Restriction enzymes
Sterile techniques
References
Chapter 2 Agarose gel electrophoresis
Definition
Rationale
Before you begin
Key resources table
Materials and equipments
Materials and equipments required
Reagents required
Step-by-step method details
Timing: 1-1.5 h (depending on the size of the sample)
Expected outcomes
Quantification and statistical analysis
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Alternative methods/procedures
References
Chapter 3 SDS-polyacrylamide gel electrophoresis
Definition
Rationale
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Timing: 3-4 h (depends on the plate size)
Expected outcomes
Quantification and statistical analysis
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Alternative methods/procedures
References
Chapter 4 Immunoelectrophoresis
Definition
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected outcomes
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
References.
Chapter 5 Plasmid DNA isolation using alkaline lysis method from E. coli DH5 α cells
Definition and rationale
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected outcomes
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Alternative methods/procedures
References
Chapter 6 Restriction digestion of plasmid DNA
Definition and rationale
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected outcomes
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
References
Chapter 7 Genomic DNA extraction from the plant leaves using the CTAB method
Definition
Rationale
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected outcomes
Quantification and statistical analysis
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Alternative methods/procedures
References
Chapter 8 Genomic DNA extraction from silkworm using modified phenol:chloroform method (Suzuki et al., 1972)
Definition and rationale
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected outcomes
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Alternative methods/procedures
References
Chapter 9 Genomic DNA extraction from Fungal Mycelium using the modified Cenis, 1992 method
Definition and rationale
Before you begin
Key resources table
Materials and equipment.
Step-by-step method details
Expected outcomes
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Alternative methods/procedures
References
Chapter 10 Bacterial transformation using CaCl 2
Definition
Rationale
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected outcomes
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Alternative methods/procedures
References
Chapter 11 Transformation of E. coli by electroporation
Definition
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected outcomes
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Alternative methods/procedures
Further reading
Chapter 12 Transfection of cells using lipofectamine
Definition
Key resources table
Materials and equipments
Step-by-step method details
Advantages
Optimization and troubleshooting
Problem
Potential solution
References
Chapter 13 Total RNA isolation
Definition and rationale
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected outcomes
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Alternative methods/procedures
References
Chapter 14 RNA isolation from plant tissues with high phenolic compounds and polysaccharides
Definition and rationale
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected outcomes.
Quantification and statistical analysis
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Further reading
Chapter 15 Total protein extraction with TCA-acetone and 2D gel electrophoresis
Definition
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected results
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Further reading
Chapter 16 Dansyl-Edman method of peptide sequencing
Definition
Key resources table
Materials and equipment
Step-by-step method details
Advantages
Limitations
Further reading
Chapter 17 ISSR analysis
Definition
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected outcomes
Advantages
Limitations
Safety considerations and standards
Alternative methods/procedures
Further reading
Chapter 18 Gene cloning/recombinant DNA technology
Definition
Rationale
Materials and equipments
Attributes of an ideal vector
Attributes of a good host cell
Step-by-step method details
Plant material
Microbial cultures and plasmids
DEPC treatment
RNA extraction and qRT-PCR
DNase treatment of RNA samples
Quantitative and qualitative analysis of RNA
cDNA synthesis
Primer designing
Validation of cDNA and primers using PCR
PCR reaction mixture
PCR cyclic conditions
Gel electrophoresis
cDNA quantification
Confirmation of cDNA synthesis
Gene cloning
PCR amplification of full-length genes (CBF-1)
Restriction digestion of the PCR fragments and vector
Expression vector
Ligation
Transformation
Confirmation of cloning by restriction digestion.
Heterologous expression
Cell culture experiments
Expected outcomes
Advantages
Limitations
References
Chapter 19 Polymerase chain reaction (PCR)
Definition
Rationale
Types of PCR
Before you begin
Key resources table
Materials and equipment
Step-by-step method details
Expected outcomes
Advantages
Limitations
Applications of PCR
Applications of PCR in agricultural sciences
Optimization and troubleshooting
Problem
Potential solution
Alternative methods/procedures
Further reading
Chapter 20 Blotting/hybridization and its types
Definition
Types of blotting
Step-by-step method details
Applications
Step-by-step method details
Applications
Step-by-step method details
Applications
Step-by-step method details
Applications
Further reading
Chapter 21 Dot blot analysis
Definition and rationale
Before you begin
Materials and equipment
Step-by-step method details
Expected outcomes
Advantages
Limitations
Further reading
Chapter 22 ELISA (enzyme-linked immunosorbent assay)
Definition
Rationale
Types of ELISA
Materials and equipment
Step-by-step method details
Quantification and statistical analysis
Advantages
Limitations
Further reading
Chapter 23 Study of principle of centrifugation
Definition
Types of centrifuge
Before you begin
Materials and equipment
Step-by-step method details
Expected outcomes
Advantages
Limitations
Optimization and troubleshooting
Problem
Potential solution
Safety considerations and standards
Alternative methods/procedures
Further reading
Chapter 24 Study of principle of chromatography
Definition
Different types of chromatographic techniques
Expected outcomes
Advantages
Limitations
Further reading.
Chapter 25 Thin layer chromatography (TLC).