Practical Boitechnology Principles and Protocols Thatoi, Hrudayanath

Contents
Preface
Acknowledgements
1. General Laboratory Principles
1.1 Safety Rules
1.2 Cleaning Glassware and Plasticware
1.3 Basic Laboratory Techniques
Review Questions
2. Biochemistry
2.1 Determination of pKa Value
2.2 Validation of Beer-Lambert Law
2.3 Determination of Absorption Maxima
2.4 Calibration Curve of Starch
2.5 Carbohydrates
2.6 Quantitative Analysis of Carbohydrates
2.7 Estimation of Proteins
2.8 Estimation of Amino Acids
2.9 Estimation of Lipid
2.10 Estimation of DNA
2.11 Estimation of RNA
2.12 Estimation of Ascorbic Acid in Plant Materials
2.13 Estimation of Thiamine
2.14 Quantitative Estimation of Phosphate Solubilisation in Culture Medium
Review Questions
3. Molecular Biology-I (Genomic Technology)
3.1 Isolation of Genomic DNA
3.2 Isolation of Plasmid DNA
3.3 Isolation of RNA
3.4 DNA Quantification and Quality Analysis
3.5 Agarose Gel Electrophoresis for DNA Quantification and Quality Analysis
3.6 Restriction Digestion
3.7 PCR Amplification
3.8 DNA Fingerprinting Technique
3.9 DNA Sequencing
3.10 Blotting Technique
3.11 Cloning
Review Questions
4. Molecular Biology-II (Proteomics Techniques)
4.1 Protein Precipitation by Salting Out Method
4.2 Dialysis
4.3 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) of Proteins
4.4 Native-PAGE
4.5 2-D Electrophoresis
Review Questions
5. Immunological Techniques
5.1 Immunodiffusion Techniques
5.2 Immunoelectrophoresis
5.3 Rocket Immunoelectrophoresis
5.4 Counter Current Immunoelectrophoresis
5.5 Enzyme-Linked Immunosorbent Assay (ELISA)
5.6 Antibody Production
5.7 Assay of Non-Specific Immune Parameters
Review Questions
6. Cytogenetics and Haematology
6.1 Study of Different Stages of Mitosis
6.2 Study of Different Stages of Meiosis
6.3 Karyotyping
6.4 Blood Grouping
6.5 Differential White Blood Cell Count (Differential Leukocyte Count)
6.6 Total Red Blood Cell Count
6.7 Estimation of Haemoglobin (Hb)
Review Questions
7. Microbial Techniques
7.1 Cleaning Glassware
7.2 Sterilization
7.3 Preparation of Culture Media
7.4 Techniques for Isolation of Pure Cultures
7.5 Gram Staining
7.6 Acid-Fast Staining
7.7 Fungal Staining
7.8 Mycorrhizal Staining
7.9 Isolation of Cyanobacteria
7.10 Isolation of Rhizobium
7.11 Microbial Growth Curve
7.12 Isolation of Bacteriophage
7.13 Isolation of Tobacco Mosaic Virus (TMV)
7.14 Antibiotic Assay
7.15 Bacterial Sensitivity
7.16 Paddy Straw Mushroom Cultivation
Review Questions
8. Fermentation and Enzyme Technology
8.1 Starter Culture Preparation for Bioethanol Production
8.2 Production of Ethanol
8.3 Ethanol Fermentation by Immobilization of Yeast
8.4 Estimation of Alcohol
8.5 Wine Production
8.6 Beer Production
8.7 Bioreactor Designing
8.8 Amylase Production
8.9 Enzymatic Assay
Review Questions
9. Plant Tissue Culture Techniques
9.1 Preparation of Culture Media
9.2 Callus Culture
9.3 Direct Organogenesis: Shoot Tip Culture
9.4 Embryo Culture
9.5 Cell Suspension Culture
9.6 Anther Culture
9.7 Induction of Hairy Roots using Agrobacterium Rhizogenes
9.8 Agrobacterium Mediated Biotransformation
9.9 Artificial Seeds/Synthetic Seeds
9.10 Protoplast Culture
9.11 Isolation of Protoplast by Mechanical Method
9.12 Isolation of Protoplast by Enzymatic Method
9.13 Gene Transfer by Particle Bombardment
9.14 Gene Transfer by Microinjection Technique
9.15 Gene Transfer by Electroporation Technique
9.16 PEG-Mediated Gene Transfer
Review Questions
10. Animal Cell Culture
10.1 Preparation of Media
10.2 Primary Cell Culture
10.3 Preparation of Established Cell Lines
10.4 Maintenance of Cell Lines
10.5 Preservation of Cell Lines
Review Questions
11. Pharmaceutical Bioprocessing Techniques
11.1 Extraction Techniques used for Isolating Phytochemicals from Medicinal Plants
11.2 Liquid-Liquid Extraction Technique
11.3 Chromatography
11.4 Qualitative Phytochemical Assay
11.5 Estimation of Total Flavonoid Content
11.6 Estimation of Total Tannin Content
11.7 Cytotoxicity Study (MTT Assay)
Review Questions
12. Analytical Procedures for Water Analysis
12.1 Dissolved Oxygen (DO)
12.2 Biochemical Oxygen Demand (BOD)
12.3 Chemical Oxygen Demand (COD)
12.4 Estimation of Phosphate (PO43–)
12.5 Estimation of Ammonia (NH3)
12.6 Estimation of SO42– (Sulphate)
12.7 Estimation of Nitrite
12.8 Estimation of Nitrate (NO3–)
12.9 Estimation of Chloride (Cl–)
12.10 Estimation of Total Hardness
12.11 Determination of Total Alkalinity (Richards, 1954)
12.12 Estimation of Total Dissolved Solid
12.13 Estimation of Hexavalent Chromium
Review Questions
13. Biostatistics
13.1 Mean, Median, Mode, Standard Deviation
13.2 T-Test
13.3 Regression and Correlation
13.4 Analysis of Variance (ANOVA)
Review Questions
14. Bioinformatics Techniques
14.1 Database Search
14.2 BLAST (Basic Local Alignment Search Tool)
14.3 FASTA
14.4 Multiple Sequence Alignment
14.5 Study of Human Genome Data
14.6 Homology Modelling
Review Questions
15. Biophysical Techniques (Bioinstrumentation)
15.1 Laminar Airflow
15.2 Centrifuge
15.3 Lyophilizer
15.4 Microscopy
15.5 UV-VIS Spectrophotometer
15.6 Electrophoresis
15.7 Gel Documentation System
15.8 UV-Transilluminators
15.9 High Performance Liquid Chromatography (HPLC)
15.10 Thermal Cycler
15.11 Microplate Reader
15.12 Infrared Spectroscopy
15.13 Nuclear Magnetic Resonance (NMR)
15.14 Mass Spectroscopy
15.15 X-Ray Diffraction
15.16 Microarray
Review Questions
Index

This book principally contains practical aspects of biotechnology, its theoretical background, working principles, and procedures meant for conducting experiments in the laboratories. The experiments in this book are spread in different chapters like Biochemistry, Microbiology, Immunology, Molecular biology, Plant and Animal Biotechnology, Environmental Technology, Industrial Biotechnology, Biostatistics and Bioinformatics. Besides, a chapter has been devoted towards discussion on safety guidelines, setting up of biotechnology laboratory, ethical issues concerning microbiology and genetic engineering work along with different reagent preparation for conducting laboratory experiments. Each chapter aims to describe both the theory and relevant practical details for a given technique, and to identify both the potential and limitations of the technique. This book is designed as an intensive introduction to the various tools of molecular biology. It introduces all the basic methods of molecular biology including cloning, PCR, Southern blotting, Northern blotting, Western blotting, DNA sequencing.
This book is useful for final year undergraduate (especially project) students, post-graduate research students and research scientists and technicians who wish to understand and use new techniques in the field of Biotechnology.

English