Analytical Techniques in Biotechnology: A Complete Laboratory Manual

Description

Cover Half Title Author's Profile Title Copyright Dedication Contents Preface Abbreviations Chapter 1: Introduction to the Laboratory Basic Pre-Laboratory Preparations Biology Lab Safety Rules Philosophical and Ethical Issues Suggestions to the Instructor General Information for Keeping a Laboratory Notebook Laboratory Reports (a) Aim or Objective (b) Basic Principle in Brief (c) Materials or Requirements (d) Methods or Procedure (e) Data Tabulation or Observation (f) Result(s) or Finding(s) (g) Figure(s) and Finding(s) (h) Discussion and Conclusion(s) (i) Special Precautions (if any) (j) References Ideal Laboratory Conditions Operation Techniques of Some Equipment and their Basic Working Principles (a) Pipetting with Pipetman (b) Measuring Absorbance with a Spectrophotometer (c) Preparing Dilutions (d) Buffer and pH Determination (e) Metric Length and Fluid Volume (f) Using a Microscope (g) Binomial Nomenclature (h) Centrifugation Technique (i) Polymerase Chain Reaction (PCR) (j) Electrophoresis Technique (k) Sterilization Techniques (l) Autoclaving (m) Distillation Chapter 2: Genetics Expt. 1 Study of various stages of Meiosis and Mitosis calculating Mitotic Index Expt. 2 Preparing Karyotype of human chromosomes from photograph Expt. 3 Problems in Mendel’s Law of Dominance, Segregation and Independent Assortment Expt. 4 C-banding technique of Zea mayz pachytene chromosomes Expt. 5 Problems in Incomplete Dominance Modifi ed Ratios and Multiple Alleles Expt. 6 Problems in Linkage Expt. 7 Problems in Sex-determination Expt. 8 Problems in Quantitative Inheritance Expt. 9 Estimation of Genetic parameters and partitioning of variance Expt. 10 Problems in Probability, Test of Signifi cance (Chi-Test) and Hardy Winberg’s Law Chapter 3: Microbiology Expt. 1 A. Isolation of bacteria from Curd, Root nodules, Soil and Water samples B. Isolation of bacterial and fungal spores from ‘Air samples’ Expt. 2 Aseptic technique and transfer of microorganisms for pure culture Expt. 3 A. Staining Gram Positive (+ve) and Gram Negative (–ve) bacteria B. Staining bacterial endospore Expt. 4 Determination of bacterial growth curves by (a) Manual counting (by microscope), and (b) UV-Spectrophotometer method Expt. 5 Identifi cation of bacteria through Biochemical test and Pigmentations Expt. 6 Uses of Physical and antimicrobial agents to control microorganisms Expt. 7 Determine which type of garbage is Bio-degradable, and which are non-degradable Expt. 8 Isolation and Identifi cation of Non-Pathogenic Fungi Expt. 9 Determine the cell viability by dye-exclusion method Expt. 10 Plaque count by infecting the bacterium Escherichia coli B with its specifi c bacteriophage Coliophage T4. (See Chapter VII; Expt. 8) Chapter 4: Plant Tissue Culture Expt. 1 Preparation of Stock solution for MS basal medium and Plant Growth Regulator Stock Expt. 2 Aseptic culture technique for establishment and maintenance of carrot root cubes and Tobacco plant by leaf disc culture Expt. 3 Aseptic organ culture technique, of embryo and ovary Expt. 4 Determine callus growth and calculate the Callusing Index Expt. 5 Study the effect of auxin or cytokinin and the plant growth regulators Expt. 6 Micro-propagation of rice by in vitro organogenesis from embryo Expt. 7 Indirect somatic embryogenesis in carrot (Daucus carrota) Expt. 8 A. In vitro germination of orchid seeds B. In vitro germination of Tobacco, and Tomato seeds Expt. 9 Exact protoplast from plant tissue and demonstrate the somatic cell fusion Expt.10 Production of synthetic seeds and test their viability Chapter 5: Biochemistry, Enzyme and Fermentation Technology Expt 1A Estimation of mouse or chicken liver glycogen by anthrone method Expt 1B Preparation of liver homogenate and estimation of liver glycogen Expt 2 Extraction of lipid from egg yolk Expt 3 Separate lipids by thin layer chromatography (TLC) method Expt 4 Protein extraction and estimation from mouse liver Expt 5 Preparation of standard curve of protein (BSA) by Folin-Lowry method and determination of unknown protein concentration Expt 6 To study the effect of enzyme lactase on milk Expt 7 To immobilize yeast cells by sodium alginate entrapment method and to study the rate of conversion of glucose by immobilized yeast cells Expt 8 Protease estimation from fruits Expt 9 Extraction of ethyl alcohol from molasses by yeast fermentation Expt 10 Sauerkraut production and to analyze certain parameters during production (the period of fermentation) Chapter 6: Cell and Molecular Biology Expt 1 Estimation of nucleic acid. To estimate the RNA isolated from a given sample OR Spectrophotometric analysis of RNA/ orcinol determination of RNA Expt 2 Estimation of DNA by diphenylamine (DPA) method. OR Preparation of DNA standard curve through spectrophotometer analysis using diphenylamine reagent Expt 3 Extraction of Genomic DNA. To extract genomic DNA from a given plant specimen by using CTAB method (Based on method of Saghai- Maroof et al., 1984) Expt 4 DNA extraction from blood Expt 5 Perform agarose gel electrophoresis for screening the DNA isolated and to estimate the relative sizes Expt 6 RNA isolation and cDNA preparation. A. To isolate total RNA from liver tissue by Trizol method B. Complimentary DNA (cDNA) preparation from the isolated total RNA Expt 7 Fractionation of cellular components- A. Isolation of mitochondria. B. Isolation of chloroplasts from a given plant specimen Expt 8 Separation of proteins by SDS gel electrophoresis Expt 9 Blood smear preparation and screening. To prepare a blood smear and to study the types of cell present Expt 10 Problems in molecular genetics Chapter 7: Recombinant DNA Technology Expt 1 Bacterial Transformation. Competent cell preparation. Bacterial transformation and screening of transformed colonies Expt 2 Isolation of plasmid. Plasmid alkaline lysis-miniprep protocol Expt 3 Detection of plasmid DNA by agarose gel electrophoresis Expt 4 Restriction digestion of DNA and its analysis by agarose gel electrophoresis Expt 5 Polymerase chain reaction—To set up a standard PCR reaction for a specifi c gene fragment of interest Expt 6 Gene cloning—To clone a gene of insert in a suitable vector system Expt 7 Bacteriophage isolation—To isolate bacteriophage/coliphages from the sewage sample Expt 8 Transformation by co-cultivation techniques in tobacco plants (leaf discs) using Agrobacterium tumefaciens Expt 9: Medical Biotechnology—To separate blood mononuclear cells by Ficoll hypaque and observing viable PBMC’s by trypan blue staining Expt 10 Bioinformatics A. To analyze a given gene sequence for mutation by software analysis B. To analyze a given protein sequence for mutation by software analysis Chapter 8: Animal Biotechnology and Immunology Expt.1 To learn the basics of animal cell culture Expt. 2 To describe the maintenance of cells in culture Expt. 3 To estimate the number of viable cells using the trypan blue viability test method Expt. 4 Protein extraction and estimation from cell lines by immuno-blotting Expt. 5 Immunology. Bacterial antigen preparation—Preparation of antigens from bacterial culture routes of inoculation of antigens in rabbit/mice by different routes of immunization Expt 6 Screening for the development or formation of the primary antibody against the inoculated (administered) antibody by- A. ‘Haemaglutination’ B. ‘Immuno-diffusion’ methods Annexures Annexure I List of Equipments required for conducting Biotechnology practical Annexure II Common Buffers usually used in Biotechnological experiments Annexure III Stains and Nutrient Media composition for Microbiological experiments Annexure IV Plant Tissue Culture Media Composition Annexure V Answers to the Problems in Chapter II: Genetics and Chapter VI: Cell and Molecular Biology Annexure VI Chi-square table (χ2 ) Annexure VII References/ Technical Literatures Annexure VIII Addresses of Some Suppliers of Equipments (Minor and Major), Chemicals, Glass- and Plastic-wares Index