Life Sciences/ Biotechnology
Laboratory Manual on Biotechnology
Description
Today, plant biotechnology has become entrenched as a branch of science, and biotechnology courses have been included not only at university but even at school level reflecting the importance of the subject. Recently biotechnology has been introduced in syllabus of most of the universities, at the graduate and/or post graduate level(s), either as a separate paper or a part of it. For the better understanding of the subject it is imperative that students should be facilitated to learn the elementary laboratory protocol on different aspects of plant biotechnology. With this end in view, authors have prepared the present book entitled, Laboratory Manual on Biotechnology. The authors have been teaching biotechnology and realizes the problems of students. Therefore, this book is compiled to provide the students, the current informations of different areas of practical biotechnology. In this book, various protocols have been described in very simple manner and worksheets have been provided to record the observations etc. Exercises have been so arranged that they have become linked to each other for better understanding of the rationale to carry out the exercises. The manual deals with seven different parts. The language of the text is simple and the subject matter is fully illustrated. In this book each experiment includes introduction, principle, materials, procedure and calculations. In addition, there are 19 appendices which give minute details on basic chemistry, buffer preparations and other aspects required for the conduct of the experiments. The methods given in the book will be useful for conducting practical classes at the undergraduate and post graduate levels in biotechnology, biochemistry, microbiology, agricultural sciences, environmental science, botany, zoology, genetics and plant breeding, pharmaceutical science and other biology-related subjects. This book will be bonanza for the research workers.
Table of Contents
Foreword, Preface, Unite-I General Laboratory Principle Laboratory, Common Laboratory apparatus, Instructions to work in laboratory, Some important precautions, Practical note book, Laboratory requirements and safety, Laboratory requirements and safety, Laboratory techniques, Use of equipments, Methods in molecular biology Unit-II Basic and Advanced (DNA, RNA and Protein) Molecular Techniques, To isolate plant DNA using Cety1 Trimethy1 Ammonium Bromide (CTAB), Isolation of bacterial genomic DNA, To perform the whole blood DNA extraction, To study the estimation of DNA by Diphenylaming (DPA) method, To perform Agarose Gel Electrophoresis (AGE), To isolate plasmid DNA from bacteria, To study the isolation of yeast genomic DNA using miniprep method, To Perform fungal DNA extraction, Quantitative determination of DNA and RNA by spectrophotometric method, To study the elution of plant DNA fragment form agarose gel, To retrieve and purify and specific DNA fragment from an agarose gel through dialysis Electro elution, to perform PCR amplification of specific target sequence from genomic DNA, To perform Miscrosatellite or Simple Sequence Repeats (SSR) analysis, To study the Southern Hybridization, To develop AFLP molecular marker for target sequence from genomic DNA, To perform the study of transformation of the desired bacterial strain with plasmid DNA through chemical transformation, To perform the study of transformation of the desired bacterial strain with plasmid DNA through electroporation, To perform the study of restriction digestion of DNA, Preparation of Phage Lysates, Preparing DNA from phage Lysates using CsCI gradient centrifugation, Preparing DNA from Phage Lysates by boiling lysis mini-prep method, Dephosphorylation of restriction enzyme digested vector pUC18, To perform the study of ligation of EcoRI digest using T4 DNA ligase, Extraction of nucleic acids and their detection, Determination of the quantity of nucleic acid by optical density method, To study the isolation of total RNA, To find out the determination of RNA by orcinol method, Isolation of plant proteins from seeds, Isolation of plant proteins from plant leaves, Preparation of muscle’s protein, Estimation of the concentration of protein in given sample by absorbance assay method, SDS-Polyacrylamide slab gel electrophoresis of proteins under reducing conditions (SDS-PAGE), To find out the max of the given test solution using spectrophotometer, To determine the concentration of given salt solution by using spectrophotometer, To perform DNA sequencing and determine the DNA sequencing by reading and autoradiogram obtained from a sequencing gel, To study Polymerase Chain Reaction Cloning, To study the working of PCR, Isolation of total plant RNA from Rice plants and Reverse Transcriptase Polymerase chain reaction Unit-III Plant Tissue Culture, To grow individual cells on a nutrients medium using various methods, Plant regeneration from callus or plant tissue, Isolation and culture of plant protoplasts, Protoplast fusion (somatic cell hybridization), Production of Pathogen-free plants through meristem culture, To demonstrate the transformation of Agrobacterium, To demonstrate the agrobacterium-mediated transformation of tobacco, To study Particle bombardment-mediated transformation of plant, Direct gene transfer to protoplasts by PEG-mediated transformation of Brassica protoplast, Direct gene transfer to protoplasts by electroporation method in tobacco plants Unit-IV Enzyme Biotechnology Determination of Nitrate reductase activity, Determination of enzyme glutamate dehydrogenase activity (GDH) from plant leaves/shoot./root, Determination of enzyme Glutamine synthetase activity (GS) from plant tissue, To estimate the catalytic power of acid phosphatase, To study the progress curve obtained during the hydrolysis of p-nitropheny1 phosphate by serum alkaline phosphate (orthophosphoric monoester phosphohydrolase,3.13.1), To study the variation of serum alkaline phosphatase activity with enzyme concentration, Estimation the amylase activity in the given sample by calculating units (U) of enzyme, Investigation the effect of temperature on amylase activity and find out the temperature optima for amylase, The determination of the Michaelis constant for the digestion of casein by trypsin (3.4.4.4.), To study the Yeast isocitrate dehydrogenase: an allosteric enzyme, Nicotionamide adenine dinucleotide as the coenzyme for lactate dehydrogenase (L-lactate: NAD oxidoreductase, 1.1.1.27), The effect of inhibitors on ox heart lactate dehydrogenase, The determination of the ‘optimum temperature’ of amylase, The effect of temperature on the stability of amylase, Unit-V Biochemistry, To learn preparation and handling of solutions, Preparation of buffer, Identify the carbohydrate present in the given sample by qualitative method, Estimate the concentrations of reducing sugar present in given sample by DNSA method, Estimate the concentration of sugar present in given sample by Anthrone method, Identification of N-terminal amino acid of a protein using dansy1 chloride method, Identify the amino acid present in the given sample by qualitative method, Estimate of concentration of amino acids in given sample by Ninhydrin method, Estimate the concentration of protein in given sample by Bluret method, Isolation of milk protein (casein) from the milk protein, Estimate the concentration of protein in a given sample by Folin-Lowry’s method, Estimate the concentration of protein in given sample by Bradford’s method, Extraction and Gravimetric estimation of lipid from group nut by Soxhlet’s method, Estimate the lipid present in given sample by Sulfo-phosphovanilin method, Estimate concentration of inorganic phosphorus present in the given sample by Fiske & Subbarrow’s method, Estimate of the concentration of DNA present in given sample by Diphenylamine (DPA) method, Separation and identification of amino acids by ascending paper chromatography (TLC), Separation of pigments from leaves or flowers by ascending paper chromatography, To demonstrate the practical aspects of Haemoglobin Electrophoresis techniques in general laboratory, To demonstrate the purification of proteoglycans by ion-exchange chromatography, To demonstrate the gel permeation chromatograplhy of proteins, To demonstrate the preparation of Affinity Matrix (Sepharose-Pepsin Conjugate), Analysis of protein by gel electrophoresis under denaturing conditions (SDS-PAGE), Determination of ascorbic acid content in plant material, Determination of vitamin C in plant material, Determination of the vitamin C content in fresh fruits, Determination of vitamin C and dehydroascor acid, To separate and characterize the anthocyanin pigments, To estimate total phenol contents in plants, Estimation of total chlorophyII and carotenoid of plant tissue, Determination of total nitrogen from fresh plant tissue, Estimation of total protein of plant fresh tissue, Estimation of total amino acid of plant fresh tissue, Unit-VI Plant physiology, Demonstration of the law of diffusion through small pores, Determination of osmotic concentration of cell sap by plasmolytic method, Determination of the osmotic value of plant tissue, Determination of the osmotic pressure of cell contents, Determination of water potential (ØW) of plant tissue (Solanum tuberosum), Determination of the permeability of living tissues, To study the water conducting tissues, To demonstrate the ascent of water in plants, To study the translocation of carbohydrates from leaves, To study the (I) Structure, (II) Size, (III) Stomatal frequency and total number of stomata on a leaf of any plant, To study different kinds of stomata, their distribution, pore size and determine the ‘Stomatal Index’, Measurement of the rates of transpiration, Measuring the effect of environmental factors on the rate of transpiration, Comparison the rate of absorption with that of transpiration, To compare the rates of transpiration from two surfaces of a leaf, Determination of the changes in stomatal opening in light, darkness and under desiccation, To determine relative turgidity in plant materials, Finding out the effect of soaking seeds in water on germination , Demonstration of the diastase activity in germinating seeds of barley, To study the tissue tests for mineral nutrients of plants, Estimation of total nitrogen by Micr-Kjeldahi method, To study iron and zinc tests, To findout the effect of gibberellic acid on the production of reducing sugars the basis for bioassay of gibberellins, To isolate the chloroplast pigments on the basis of their differential solubility in various organic solvents and their absorption properties using paper chromatography, Quantitative determination and study of absorption spectrum of chlorophylls extracted from the leaves of higher plants, Isolation of active chloroplasts from leaves and observation of Hill reaction activity of chloroplasts, Estimation of stomatal and mesophy11 limitations of photosynthesis, Estimation of stomatal and assimilation loop gains, Determination of in vitro carboxylation efficiency (a/ci) based on CO2 response curves, Photorespiration based on ‘tps’ and ‘aps’ measurements, Quantification of RuBisCO by ELISA, An approach to identify high CER and CGR types form germplasm lines, To study of the method to measure maintenance of respiration, To separate anthocyanins by paper and/or thinlayer chromatography, To separate and identify the lycopene present in plant material, To colour development in apples under the influence of quality of light, To estimate the total phosphorus in plant tissue UNIT-VII Environmental Biotechnology, To determine the amount of dissolved oxygen in waste water by iodometric titration method, To determine the Biochemical Oxygen Demand (BOD) of a given waste water sample, To determine total, temporary (carbonate), permanent (non-carbonate) hardness of water sample by complex metric titration (EDTA) method, To determine total solids (TS), total dissolved solids (TDS) and total suspended solids (TSS) in a given water sample, To determine the total alkalinity of the given sample of water using standard acid solution, To determine the acidity of a waste water sample, To determine the concentration of hexavalent chromium in the given water samplet, To determine the amount of nitrate in waste water by Spectrophoteometric method, To determine the amount of sulphate ion in the sample by Nephelometrc method, To conduct jar tests on a sample of natural surface water in order to estimate an optimum dosage of ferric chloride for removal of suspended matter Unit-VIII Genetics and Cytogenetics, To study the life cycle of genetically important living organism, Study of the taxonomy and floral structure in pea, To study the exasculation, pollination (crossing) in Pea, To study the somatic and gametic chromosomes no. of common plants and animals, To study Mitosis and Meiosis in plant cell, To study Mendelian inheritance, Solving the different numerical problems of gamete formation in monohybrid, dihybrid crosses, To study the trihybrid cross using Farked line method, To determine the unknown genotype, To study detection of linkage, To study probability determination of and event, To examine the goodness of fit using chi square test, To study the Linkage and crossing over using THREE POINT TEST CROSS, To study the linkage in fungi, To study the Population Genetic (gene and genotype frequency) Suggested Reading, Appendices, Glossary, Essential Equipments for Laboratory, Index