Protocols used in Molecular Biology

Description

Protocols used in Molecular Biology is a compilation of several examples of molecular biology protocols. Each example is presented with a concise introduction, materials and chemicals required, a step-by-step procedure and troubleshooting tips. Information about the applications of the protocols is also provided. The techniques included in this book are essential to research in the fields of proteomics, genomics, cell culture, epigenetic modification and structural biology. The protocols can also be used by clinical researchers (neuroscientists and oncologists, for example) for medical applications (diagnostics, therapeutics and multidisciplinary projects). Cover 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 Back Cover