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