Analytical Techniques in Biotechnology: A Complete Laboratory Manual

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