Advances in Molecular Techniques

Chapter 1: DNA Manipulative and Ingratiative Techniques
Isolation of Total Genomic DNA
Isolation of Plant DNA Using the CTAB Method
Introduction
Experimental Outline
Materials Required
Pre-Lab Preparation
Isolation of Plant DNA Using the SDS Method
Introduction
Materials Required
Method
Precautions
To Isolate Plasmid DNA from Bacteria
Introduction
Materials Required
Method
Precautions
Isolation of Chloroplasts
Introduction
Experimental Outline
Materials Required
Pre-Lab Preparation
Method
Results
Precautions
Isolation of Chloroplast DNA
Introduction
Experimental Outline
Materials Required
Pre-Lab Preparation
Method
Results
Precautions
Isolation of Yeast Genomic DNA
Introduction
Materials Required
Method
Isolation of Bacterial Genomic DNA
Introduction
Materials Required
Method
Large-Scale Isolation of Cosmid DNA
Isolation
Purification
Miniprep Double-Stranded DNA Isolation
Principle
Isolation
Standard Alkaline Lysis Purification
Diatomaceous Earth-Base Purification
Plasmid Miniprep by Birnboim Method
Materials Required
Plasmid Miniprep by Boiling Method
Materials Required
Method
Isolation of Lambda (λ) DNA
Materials Required
Method
Quick Method for isolation of Lambda (λ) DNA
Materials Required
Single-Stranded M13 DNA Isolation Using Phenol
Method
Biomek-Automated Modified-Eperon IsolationProcedure for Single-Stranded M13 DNA
Method
Isolation of Aspergillus DNA
Materials Required
Method
Isolation of Aspergillus DNA by the Qiagen Method
Materials Required
Method
Purification of Genomic DNA
Introduction
Principle
Materials Required
Method
Materials Required
Isolation of RNA from Plant Tissues
Introduction
Extraction of Total RNA
Materials Required
Method
Precautions
Extraction of Polysomal RNA of Nuclear Sequences
Materials Required
Chemicals
Glassware and Other
Equipment
Isolation of Total RNA form Bacterial Cells
Introduction
Materials Required
Method
Quantitative Determination of DNA and RNA by a Spectrophotometric Method
Introduction
Materials Required
Agarose Gel Electrophoresis
Introduction
Preparation of Agarose Gel
Electrophoresis of DNA Fragments
Visualization of DNA Fragments
Materials Required
Chemicals
Glassware and Other
Equipment
Electrophoresis of DNA: Linear, Circular, and Super Coiled
Introduction
Experimental Outline
Materials Required
Method
Precautions
Elution of Plant DNA Fragment from Agarose Gel
Introduction
Materials Required
Method
Precautions
Hybridization and Autoradiography of DNA
Introduction
Experimental Outline
Materials Required
Method
Hybridization
Autoradiography
Precautions
PCR in Outline
Introduction
Choice Between Cloning and PCR
Experimental Outline
Materials Required
Method
Precautions
PCR Amplification of Specific Target Sequence from Genomic DNA
PCR Consist of 3 Basic Steps
Materials Required
Method
Gel Analysis
Precautions
Isolation of Total Plant RNA from Rice Plants and Reverse Transcriptase Polymerase Chain Reaction
Introduction
Nuclear Genes
Necessary Conditions for Reaction
Materials Required
Pre-Lab Preparation
Method
Reverse Transcriptase-Polymerase Chain Reaction
Precautions
Isolation of Gene of Interest by PCR
Isolation of Genomic DNA
Materials Required
Chemicals
Glassware and Other
Equipment
Precautions
Isolation of pUC18 Plasmid from TOP10 pUC18 E. coli cells
Materials Required
Chemicals
Glassware and Other
Equipment
Method
Precautions
Transformation of the Desired Bacterial Strain with Plasmid DNA
Materials Required
Chemicals
Glassware and Other
Equipment
Pre-Lab Preparation
Method
Preparation of Competent Cells
Purifying pUC18/Hind III/EcoRI Digest by Gel Elution
Introduction
Materials Required
Chemicals
Glassware and Other
Equipment
Precautions
Dephosphorylation of Restriction-Enzyme-Digested Vector pUC18
Introduction
Materials Required
Method
Precautions
Restriction Digestion of pUC 18 and A-DNA
Materials Required
Chemicals
Glassware and Other
Equipment
Method
Precautions
Enzyme Linked Immunosorbent Assay (ELISA)
DAS-ELISA
DAC-ELISA
PAC-ELISA
Materials Required
Chemicals
Pre-Lab Preparation
Procedure for DAC-ELISA (Transgenic Rice)
DAC-ELISA
Precautions
Electrophoretic Separation of Plasma Protein
Materials Required
Chemicals
Glassware
Equipment
Method
Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis
Materials Required
Chemicals
Glassware and Other
Equipment
Pre-Lab Preparation
Preparation of the Separating Gel
Preparation of the Stacking Gel
Loading and Running the Gels
Staining and Destaining the Gels
Method
Production of Antibodies from Laboratory Animals
Preparation of Immunizing Material
Protein Solution
Use of Adjuvants
Alum Precipitation
Oil in Water
Inoculation and Bleeding of Animals
Purification of Enzymes for Immobilization
Equipment
Method
Purification of Hemoglobin
Purification of Fungal α-Amylase
Purification of Protease
Enzyme Immobilization in Polyacrylamide Gel
Equipment
Materials Required
Chemicals
Materials Required
Chemicals
Equipment
Chapter 2: Regulative Techniques
Restriction Fragment Length Polymorphism (RFLP)
Experimental Outline
Materials and Pre-Lab Preparations
Method
DNA Fragment Separation on Agarose Gel
Hybridization
Southern Blotting Procedure
Probe Labelling
Autoradiography
Amplified Fragment Length Polymorphism
Summary of the AFLP Analysis System
Restriction Endonucleases Digestion
Ligation of Adapter
Amplification Reaction
Method
Additional Materials Required
Restriction Digestion of Genomic DNA
DNA Blotting Techniques
Precautions
Experimental Outline
Materials
Pre-Lab Preparations
Method
Results
Discussion
RNA Separation of Species by Ion Exchange Column Chromatography on Methylated Albumin Kiesselguher Columns (MAK)
Experimental Outline
Materials
Pre-Lab Preparations
Method
Preparation of Radio-Labelled Probe by Random Primer Method
Experimental Outlines
Materials
Pre-Lab Preparations
Method
Precautions
Detection of a Specific Fragment of Plant DNA from Restriction Digest by Hybridization with a Labelled Probe
Experimental Outline
Materials
Pre-Lab Preparations
Method
Hybridization
Autoradiography
Precautions
In Vitro Transcriptions
Experimental Outline
Materials
Enzymes and Buffers
Equipment
Method
To Purify RNA by Ethanol Precipitation
Troubleshooting
In vitro Translation of mRNA
Experimental Outline
Materials
Procedure
Prepare the mRNA by in vitro Transcription
Prepare the Protein by in vitro Translation
Reagents and Solutions
Reticulocytes and Their Use in the Study of Protein Synthesis
Experimental Outline
Materials and Pre-Lab Preparation
Method
Precautions
Protein Synthesis in Intact Rabbit Reticulocytes
Experimental Outline
Materials and Pre-Lab Preparations
Method
Calculations
Precautions
Metabolic Labelling of Proteins and Immunoprecipitation
Experimental Outline
Materials and Other
Equipment
Method
Western Blots
Precautions
Marker-Assisted Pyramiding of Leaf Rust Resistance Gene in Wheat
Method
PCR Reactions
STS Marker-Based Detection of 1B/1R Translocation
Reagents
Method
STS Marker-Based Detection of Lr 26 Gene
Reagents
Method
Microsatellite Markers
Advantages of Microsatellite Markers
Applications
Method
Inter-Simple Sequence Repeat (ISSR)
Reaction Mixture for ISSR
Amplification
Isolation of Specific Full-Length cDNAs By RT-PCR Method
Isolation or RNAs by TRIzol Method
Design and Synthesis of Specific Forward and Reverse Primers
Amplification of cDNA of Interest by RT-PCR
Purification Products by High-Speed Centrifugation of Agarose Gel Slices of PCR
High Yield and Cleaner Elution of DNA
Construction of Genetic Linkage Maps and the Mapping of Quantitative Trait Loci (QTLs)
Mapmaker for the Construction of the Genetic Map, Plabstat for the Analysis of Phenotypic Data, PlaybQTL for the Interval Mapping QTL Analysis
Transposons and Their Mutagenesis
Site-Specific Transposon (Tn5) Mutagenesis
Strain
Generalized Transposon (Tn5) Mutagenesis
Requirements
Medium Composition
Preparation of Antibiotic Stock Solution
Procedure
On 2-Dimensional Gel Electrophoresis for Analysis of Gene Expression
Sample Preparation
Equipment and Reagents
Method
Rehydration of IPG Strips
Equipment and Reagents
Method
Isoelectric Focusing
Equipment and Reagents
Method
Equilibration of Isoelectric Focusing Strip before Transfer to SDS-PAGE
Equipment and Reagents
Method
Casting the 2-Dimensional Gel
Equipment and Reagents
Method
Drying of Polyacrylamide Gels
Equipment and Reagents
Method
Metabolic Labeling for Detection of Differential Gene Expression
Sample Preparation
Equipment and Reagents
Method
Preparation of Calibration Strip for Quantitation of (35S) Methionine-Labelled Spots
Equipment and Reagents
Method
Silver Staining for Mass Spectrometry
Reagents
Method
Chapter 3: Recombinant Techniques
Digestion of Plasmid DNA with Various Restriction Endonuleases and Separation of Products in Agarose Gels
Plasmids
pBluescript KS(+)
Experimental Outline
Materials and Pre-Lab Preparations
Method
Restriction Mapping
Decontamination of EtBr-Containing Solutions
Directional Cloning into Plasmid Vectors
Experimental Outline
Materials and Pre-Lab Preparations
Buffers and Solutions
Enzymes and Buffers
Gels
Nucleic Acid and Oligonucleotides
Equipment
Method
Precautions
Cloning of the Phagemid Vectors
Improvements in Phagemids and Helper Viruses
Experimental Outline
Materials
Buffers and Solutions
Gel
Media
Special Equipment
Vectors and Bacterial Strains
E. coli F′ Strain
E. coli Strain DH 115
Method
Preparation of a High-Titer Stock of Helper Bacteriophage
Growth of Recombinant Phagemids with Helper Virus
Estimation of the Yield of Single- Stranded Phagemid DNA by Gel Electrophoresis
Precautions
Gene Expression in E. coli and Analysis of Gene Products
Materials and Pre-Lab Preparations
Buffers and Solutions
Ethyl Acetate
Lysis Buffer
Nucleic Acids and Oligonucleotides
Media
Equipment
Vectors and Bacterial Strains
Method
Hybridization of the RNA to Immobilized DNA
Hybridization Controls and Troubleshooting
Precautions
Assay of the Reported Cat Gene
Reporter Genes Used in Analysis of Regulator Elements
Experimental Outline
Materials and Pre-Lab Preparations
Buffers and Solutions
Ethyl Acetate
Lysis Buffer
Special Equipment
Cells and Tissues
Lysis of Cells by Repeated Freezing and Thawing
Lysis of Cells Using Detergent-Containing Buffers
Detection of CAT Activity Using Thin-Layer Chromatography
Assay for β-Galactosidase in Extracts of Mammalian Cells
Experimental Outline
Materials and Pre-Lab Preparations
Buffers and Solutions
β-Galactosidase Substrates
Method
Precautions
DNA Sequencing
Determination of Nucleotide Sequence of DNA by Dideoxy Chain Termination Method
Experimental Outline
Materials and Pre-Lab Preparations
Method
Sequencing Reaction
Electrophoresis
Autoradiography
Hybridization Analysis of YAC Library Filters
Equipment and Reagents
Method
Preparation and Gel Purification of YAC Chromosomal DNA
Equipment and Reagents
Method
Preparation of Intact Yeast Chromosomes
Gel Purification of YAC Clones to be Used as Hybridization Probes
Construction of BAC Libraries
Equipment and Reagents
Methods
Preparation of the Vector
Preparation of the Insert DNA
Calibration of Restriction Endonuclease for Partial Digestion of the DNA Preparation
Size Fractionation and Purification of the Insert DNA
Ligation, Electroporation, and Arraying the Library
Preparation of Colony Filters for Hybridization Analysis
Equipment and Reagents
Method
Hybridization Analysis of Colony Filters
Equipment and Reagents
Method
Construction of a Binary Cosmid Library from Arabidopsis Genomic DNA
Equipment and Reagents
Method
Calibration of Restriction Endonuclease for Partial Digestion of Plant Genomic DNA
Large-Scale Partial Digestion of Genomic DNA
Treatment of Partially Digested Genomic DNA with CIAP
Size Fractionation and Purification of Genomic DNA
Preparation of Vector
Ligation of Vector and Insert DNA
Packaging and Plating the Library
Agrobacterium: Mediate Transformation
Explants, Equipment, and Reagents
Callus Induction from Immature Embryos
Agrobacterium Culture for Transformation
Transformation and Regeneration
Method
Growth of Donor Plants
Establishment of Embryogenic Calli from Immature Embryos
Preparation of Agrobacterium Culture
Transformation
Washing and Selection
Regeneration
Biolistic Transformation
Explants, Equipment, and Reagents
Methods
Preparation of Immature Embryos/Embryogenic Calli
Sterilization of Consumables and Preparation of Micro-Carrier Particles
DNA Precipitation
Operation of Particle Delivery System
Selection and Regeneration
Protoplast Transformation Using PEG
Explant, Equipment, and Reagents
Mature/Immature Embryo-Derived Calli
Establishment of Suspension Culture
Protoplast Isolation, Transformation, and Regeneration
Method
Establishment of Suspension Culture
Protoplast Isolation
Transformation Using PEG
Selection
Regeneration
Agrobacterium: Mediated Transformation of Arabidopsis Root Explants
Equipment and Reagents
Method
Preparation of Plant Material
Growth of Agrobacterium
Selection of Transformed Plants
Establishment and Transformation of Arabidopsis Cell Suspensions
Equipment and Reagents
Method
Initiation of Arabidopsis Cell Suspension
Subculture of Cell Suspension
Transformation of Cell Suspension with Agrobacterium
In Planta Transformation of Arabidopsis by Infiltration
Equipment and Reagents
Method
Growth of Plant Material
Growth of Agrobacterium
Vacuum Infiltration
Collection of Seeds
Seed Sterilization
Selection of Transformants
PCR Amplification of T-DNA Tagged Plant DNA Fragments for Automatic DNA Sequencing
Equipment and Reagents
Method
DNA Preparation
DNA Digestion
Ligation of DNA
Design of PCR Primers
Polymerase Chain Reactions
Sequence Analysis
DNA Transfection Mediated by Lipofection
Chemicals
Lipofection Reagent
Equipment
Method
Calcium-Phosphate Mediated Transfection of Eukaryotic Cells
Chemicals and Reagents
2 × HEPES Buffered Saline
Nucleic Acids and Oligonucleotides
Equipment
Method
Treatment of Cells with Chloroquine
Treatment of Cells with Glycerol
Treatment of Cells with Sodium Butyrate
Transfection Mediated by DEAE-Dextran
Materials Required
Chemicals
Equipment
Method
Transfection of DNA by Electroporation
Materials Required
Chemicals Required
Equipment
Method
DNA Transfection Method Using Polybrene
Materials Required
Chemicals
Equipment
Method
Hybridization Techniques
Nucleic Acid Hybridization
Blotting
Experimental Outline
Materials
Method
Precautions
Northern Hybridization of RNA Fractionated by Agarose Formaldehyde Gel Electrophoresis
Experimental Outline
Materials Required
Method
Precautions
Southern Hybridization of DNA Fractionated by Agarose Formaldehyde Gel Electrophoresis
Materials Required
Pre-Lab Preparations
Method
Precautions
Western Hybridization of Protein Fractionated by Agarose Formaldehyde Gel Electrophoresis
Materials Required
Protein Detection by the Use of Antibodies
Anti-Phosphotyrosine Western Blotting
Materials Required
Chapter 4: Nanobiotechnology and Metabolome Profiling
Objective: To Perform Metabolome Profiling by GC/MS Analysis
Principle
Materials Required
Chemicals
Glassware and Other
Equipment
Pre-Lab Preparations
Method
Without Solvent
With Solvent
Sample Analysis
Data Analysis
Objective: To Analyze the Presence of Known Metabolites in Plant Extract by HPLC
Principle
Materials Required
Chemicals
Glassware and Other
Equipment
Method
Preparation of Standard
Preparation of Sample
Preparation of Mobile Phase
Instrumentation and Chromatographic Conditions
Quantification of Components
Objective: To Perform Thin Layer Chromatography (TLC) for Metabolome Profiling
Principle
Requirements
Procedure
Objective: To Synthesize and Characterization of Silver Nano-Particles
Principle
Requirements
Stock Solutions
Method
Objective: To Study the Effect of Silver Nanoparticles on Plant MAPK Expression
Principle
Requirements
Method
RNA Extraction
Objective: To Demonstrate Competitive Format Based Immuno-Chromatographic Strip Using Gold Nanoparticles for On-Site Detection of Teliospores of Karnal Bunt (Tilletiaindica) in Plant
Principle
Requirements
Procedure
Metabolic Profiling Using Body Fluids by NMR Spectroscopy
Principle
Method
Appendix
Glossary
Bibliography
Index