Protocols used in Molecular Biology

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Title
Copyright
End User License Agreement
Contents
Foreword
Preface
List of Contributors
Isolation of Genomic DNA From Plant Tissues
Pallavi Singh*
INTRODUCTION
Principle
Sample Collection and Storage Conditions
CTAB DNA Extraction Buffer: 100 ml
T10E1 Buffer
DNA Isolation Protocol
Purification
Useful Tips and Suggestions for Obtaining Optimum Results
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
RNA Isolation Protocol from Cells and Tissues
Pallavi Singh*
INTRODUCTION
Precautions to be Taken Before the Experiment
I. Isolation of RNA Using TRIzol Method
Principle
Materials Required
Steps
II. Isolation of RNA Using the CTAB Method
Materials Required
Steps
III. Hot-Phenol RNA Extraction Method
Materials Required
Steps
IV. QIAGEN RNeasy Kit Method
DNase Treatment
Storage of RNA
Determining RNA Quality and Quantity
Formaldehyde Agarose Gel Electrophoresis
Materials Required
Preparation of Formaldehyde Agarose Gel
Preparation of RNA Samples for Electrophoresis [3]
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Analyzing Gene Expression through Real Time PCR while Neo-tissue Regeneration using Deve- loped Tissue Constructs
Divakar Singh1, Tarun Minocha2, Satyavrat Tripathi1, Rupika Sinha1, Shubhankar Anand1, Hareram Birla3, Vivek Kumar Pandey4, Arun Rawat3, Smita Gupta3, Sanjeev Kumar Yadav2, Pawan Kumar Dubey4 and Pradeep Srivastava1,*
INTRODUCTION
BASICS OF RT-PCR
Merits of RT-PCR
PCR Cycling
RT-PCR Master Mix Components
Thermostable DNA Polymerase
Deoxyribonucleotidetriphosphates (dNTPs)
Divalent Cation Especially Magnesium Ions
Template
RT-PCR Primer Design
Primer Designing
Basics of Primer Designing
RT-PCR Fluorescence Detection Chemistry
SYBR Green-based Detection
Mechanism of Action
Advantages
Limitations
TAQMAN probe Based Detection
Mechanism of Action
Advantages
Limitations
RT-PCR Experimental Design and Gene Expression Profiling
Setting up a Reaction Mixture
Basic PCR Protocol and Establishment of Reaction Cycle
Application of RT-PCR
CHALLENGES
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
A Modified Western Blot Protocol for Enhanced Sensitivity in the Detection of a Tissue Protein
Sachchida Nand Rai, Mallikarjuna Rao Gedda, Walia Zahra, Hareram Birla, Saumitra Sen Singh, Payal Singh, Neeraj Tiwari, Rakesh K. Singh and Surya Pratap Singh*
INTRODUCTION
SAMPLE PREPARATION
GEL ELECTROPHORESIS
BLOTTING
WASHING, BLOCKING AND ANTIBODY INCUBATION
QUANTIFICATION
TROUBLESHOOTING
CONCLUSION
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Immunohistochemistry as an Important Technique in Experimental and Clinical Practices
Hareram Birla1, Sachchida Nand Rai1, Saumitra Sen Singh1, Walia Zahra1, Neeraj Tiwari1, Aijaz A. Naik2, Anamika Misra3, Shikha Bharati4 and Surya Pratap Singh1,*
INTRODUCTION
DIRECT TECHNIQUE
NEW DIRECT TECHNIQUE (ENHANCED POLYMER ONE STEPS TRAINING METHOD)
TWO-STEP INDIRECT TECHNIQUE
POLYMER CHAIN TWO STEP INDIRECT TECHNIQUE
UNLABELED ANTIBODY ENZYME COMPLEX TECHNIQUES (PAP AND APAP)
IMMUNOGOLD SILVER STAINING TECHNIQUE (IGSS)
THE PRINCIPLE OF IHC
TISSUE PREPARATION
ANTIGEN RETRIEVAL
A. Proteolytic Enzyme Digestion
B. Heat Mediated Antigen Retrieval Techniques
ANTIGEN-ANTIBODY INTERACTION
AVIDIN–BIOTIN COMPLEX
HAPTEN LABELLING TECHNIQUE
DETECTION METHODS
QUANTIFICATION OF THE IMMUNOHISTOCHEMICAL STAINING
APPLICATION AND IMPORTANCE
TROUBLES AND LIMITATIONS
TROUBLESHOOTING OF THE EXPERIMENTAL PROBLEMS
IMMUNOENZYMATIC LABELLING PROTOCOL FOR LOCALIZA- TION OF ASTROCYTES USING ANTI-GFAP ANTIBODY
FOR CRYOSTAT SECTIONS DAY 1 PROCESSING
DAY 2 PROCESSING
IMMUNOHISTOCHEMICAL PROTOCOL FOR LABELLING ASTRO- CYTES USING ANTI-GFAP BY IMMUNE-FLUORESCENCE METHOD
FOR CRYOSTAT SECTIONS DAY 1 PROCESSING
DAY 2 PROCESSING
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Protocols for the Detection and Proteome Analysis of the Yellow Mosaic Virus Infected Soyabean Leaves
Bapatla Kesava Pavan Kumar and Surapathrudu Kanakala*
INTRODUCTION
METHOD
Protein Extraction and Virus Detection
Iso-electric Focusing
SDS-PAGE/ 2nd Dimension
Colloidal Coomassie Staining
Image Analysis
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
ABBREVIATIONS
REFERENCES
2D-DIGE A Powerful Tool for Proteome Analysis
Sudhir K. Shekhar1, Jai Godheja2 and Dinesh Raj Modi1,*
INTRODUCTION
Methodology
Steps of the Experiment
ADVANTAGES OF DIGE
Applications of DIGE
CONCLUSION
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Molecular Techniques for Genotyping
Shalini Gupta1,*, Somali Sanyal2, Suresh Kumar Yadav2 and Madan Lal Brahma Bhatt3
INTRODUCTION
1. Saliva Collection and Storage
2. Preparation for Cell Lysis
3. RNA Removal
4. Protein and Lipid Removal
5. Isolation and Purification of Genomic DNA
6. Rehydration of g DNA
[2] Several Protocols Have Been Developed to Isolate DNA from Saliva
Sample Collection
DNA Extraction
DNA Evaluation
Methods for DNA Extraction from Whole Blood
Two-step Lysis
One-step Lysis
Isolating Genomic DNA Using Magnetic Beads
GENOMIC DNA ISOLATION OF DNA FROM BLOOD
Reagents Required
Procedure
VISUALIZATION OF DNA THROUGH AGAROSE GEL ELECTRO- PHORESIS
Materials Required
METHOD
Range of separation of linear DNA of different get concentrations
Reagents
6x Gel-loading Buffer
DNA Staining Solution (10mg/ml)
EDTA (0.5M pH 8)
TAE (50X)
TBE (5X)
TPE (10X)
POLYACRYLAMIDE GEL ELECTROPHORESIS
Materials
Buffers and Solutions
METHOD
POLYMERASE CHAIN REACTION
Materials Required
Method
RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP)
Reagents Required
Method
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Sodium Bisulfite Conversion of Human Genome for DNA Methylation Studies
Aastha Mishra* and Qadar Pasha
INTRODUCTION
Mapping Whole Genome DNA Methylation Using Sodium Bisulfite Conversion of DNA Followed by Next-generation Sequencing
Sodium Bisulfite Conversion of DNA
Bisulfite Conversion Protocol
CONCLUDING REMARKS
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Chromatin Immunoprecipitation (ChIP)
Kavyanjali Sharma1, Subash Chandra Sonkar2 and Shakuntala Mahilkar*, 3
OVERVIEW OF CHIP ASSAY
GENERAL DESCRIPTION OF CHIP PROTOCOL
1. Cross-Linking
2. Cell Lysis
3. Shearing of DNA
4. Immunoprecipitation
5. Reversal of Cross-links and Characterization of DNA
6. Analysis of DNA
Requirements
Troubleshooting
Limitation
Variation
APPLICATION
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Osteosarcoma Cell Culture and Maintenance to Detect the Apoptotic Effect of Some Promising Compounds by Potent Markers viz. DNA Fragmentation and Caspase-3 Activation
Asif Jafri*, Juhi Rais, Sudhir Kumar and Md Arshad*
INTRODUCTION
Osteosarcoma Cells Culture and Maintenance
DNA Fragmentation
Detection of Apoptosis via Caspase-3 Activity in Cancer Cells
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Culture and Maintenance of Human Ovarian Carcinoma Cells for Scrutinizing Anti-cancerous Activities of Various Compounds via Some Potent Molecular Markers
Juhi Rais*, Asif Jafri, Madhu Tripathi and Md Arshad*
INTRODUCTION
I). In Vitro Culture and Maintenance of Ovarian Carcinoma Cells
II). Cell Cycle Analysis
III). Detection of Apoptosis in Cell Culture System via Annexin V- FITC
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Dictyostelium Discoideum: Live Cell Imaging in Changing Perspective
Abhishek Singh*
AN INTRODUCTION TO DICTYOSTELIUM DISCOIDEUM
The Life Cycle of D. Discoideum
Advantages of Dictyostelium Discoideum as a Model System
Required Materials
Growing of Dictyostelium Discoideumon a Bacterial Lawn
Required Materials
Recombinant Plasmid Construction
Transformation of Dictyostelium Discoideum Cells by Electroporation (Based on [15])
Materials for the Transformation of Dictyostelium Discoideum
Live Cell Imaging Microscopy
Live Cell Imaging Microscopy of Dictyostelium Discoideum Amoebic Cells (Adapted from [8])
Instrument Design of Microscope, Stage and Control of the Environment
Spinning Disk Confocal Scanner and Illumination
Imaging of GFP-labeled Fusion Proteins in Live Single Cells
Materials
Visualize Growing Cells
Evaluation of Data
DISCUSSION
Medium and Buffers
Electroporation Buffer E50
Freezing Storage Medium
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
The Recent Advancement in Rapid Golgi Method and Result Interpretation
Surya Prakash Pandey1,*, Mallikarjuna Rao Gedda2 and Abhishek Pathak1
INTRODUCTION
USE
GENERAL PRINCIPLES OF THE GOLGI METHODS
COMPOSITION OF RAPID-GOLGI FIXATIVE
RAPID GOLGI STAINING FOR SMALLER BRAIN TISSUE OR CORTICAL BRAIN
MODIFIED RAPID GOLGI STAINING SUBCORTICAL REGIONS (HIPPOCAMPUS)
MICROSCOPY Z STACKING
SHOLL ANALYSIS
MANUAL COUNTING OF SPINE AND DENDRITE
PRECAUTION
TRANSCARDIAL PERFUSION
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Molecular Markers for the Evaluation of Clonal Fidelity in Medicinal Plants
Arpan Modi and Surapathrudu Kanakala*
INTRODUCTION
MICROPROPAGATION PROTOCOL
MOLECULAR MARKERS
CONCLUSION
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Subject Index
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