Preface Biotechnology is a developing branch of science. It makes use of all basic science for industrial, agricultural and medical level applications. In this Laboratory manual almost all areas of basic Biotechnology are included. It has eight different sections with experimental protocols. Protocols are given in a simplified manner and easy to perform. General information about a Biotechnology laboratory is available in this book. Hope this will serve as a first hand laboratory manual for graduate, postgraduate and research students. Areas covered in this book include Biochemistry, Microbiology, Enzymology, Plant Biotechnology, Animal Biotechnology, Molecular biology and Genetic engineering. Protocols given in this manual are of basic experiments. Even though it is easy to perform, I request all those who do the experiments will make sure to take maximum care for the best results. Results may vary according to the laboratory conditions, quality of chemicals etc. So a careful standardization is required for good results. It is necessary to follow all general laboratory precautions before conducting all experiments.

1.     Length and Size 2.     Volume 3.     Weight

1.     Concentration

1.     General Precautions      2.     General Requirements for Biotechnology Laboratory

1.     Equipments

1.     Centrifuge      2.     Spectrophotometer      3.     Microscope

1.     Common Solvents      2.     Common Buffer Compositions

1.     Determination of lmax      2.     Verification of Beer-Lambert Law      3.     Estimation of Carbohydrate by Anthrone Method      4.     Estimation of Reducing Sugar by Di Nitro Salicylic (DNS) Acid      5.     Estimation of Protein by Biuret Method      6.     Estimation of Protein by Lowry’s Method      7.     Estimation of Protein by Bradford Method      8.     Estimation of Cholesterol      9.     Estimation of DNA      10.     Estimation of RNA      11.     Determination of iso Electric Point of Amino Acid

1.     Chromatography      2.     Identification of Sugar in Fruit Juice by Thin Layer Chromatography      3.     SDS-PAGE      4.     Composition for Native PAGE      5.     Silver Staining of Protein Gels

Enzymes are biocatalysts composed of amino acids. Enzyme reactions are highly specific. It binds to the substrate to form a reversible intermediate product called enzyme substrate complex. As the concentration of the substrate increases the rate of formation of the intermediate complex also increases. This increase in velocity of the reaction is in a hyperbolic manner till it achieves the maximum velocity called Vmax .At this point all the enzymes will be in the form of ES complex. Substrate concentration will be in at its optimum level i.e. further increase in substrate concentration will not affect the rate of the reaction. The substrate concentration at which the rate of the reaction is half the maximum velocity Vmax is called the Michaelis-Menten constant, Km. This is constant for the given enzyme acting on the same substrate.

1.     Asssay for Catalase      2.     Assay for Peroxidase      3.     Assay for Poly Phenol Oxidase      4.     Assay for Phosphatases

1.     Extraction of b-galactosidase from Yeast Cells      2.     Measurement of Enzyme Activity      3.     Immobilization of Enzymes      4.     Precipitation of Enzyme and Proteins      5.     Protien Purification by Dialysis

1.     Rules and Regulation for Safety in a Microbiology Laboratory      2.     Cleaning of Glass Wares      3.     Sterilization

1.     Preparation of Liquid and Solid Media      2.     Isolation of Pure Culture      3.     Analysis Bacterial Growth Curve      4.     Study of Effect of Carbon Source on Bacterial Growth      5.     Gram Staining      6.     Aseptic Cell Transfers      7.     Prokaryote Cell Number by Dilution Plating      8.     Cell Mass by Measurement of Turbidity      9.     Isolation of Bactriophage from Sewage Sample      10.     Standard Plate Count      11.     Imvic Test      12.     Antibiotic Sensitivity Test on Microbes      13.     Study of Transformation In Bacteria      14.     Study of Bacterial Conjugation      15.     Effect of pH and Temperature on Bacterial Growth      16.     Immobilization of Yeast Cells and Alcohol Production      17.     Citric Acid Production using Aspergillus Niger      18.     Alcohol Production from Yeast and Estimation of Alcohol

1.     Plant Tissue Culture Laboratory      2.     Cleaning and Sterilization of Glass Wares      3.     Media Sterilization      4.     Media Composition and Media Preparation      5.     Callus Culture in Carrot      6.     Culture of Shoot Tip (Vanilla)      7.     Anther Culture of Datura      8.     Subculture of Carrot Callus      9.     Hardening of Plantlets      10.     Isolation of Protoplast from Plant Leaves

1.     Isolation of Plant DNA      2.     RAPD Analysis of Genomic DNA      3.    Agro Bacterium based Transformation in Plants      4.     Assay for Gus Operon      5.     Confirmation of Transformation using Polymerized Chain Reaction (PCR)      6.     Primer Designing      7.     Isolation, Purification and Identification of Plant Secondary Metabolites      8.     Evaluation of Antioxidant Activity of Plant Extract      9.     Estimation of Total Phenolic Content in A Plant Extract      10.     Estimation of Total Flavanoid Content

1.     Animal Biotechnology Laboratory      2.    Growing Cells with Serum-Basal Medium Eagle (BME), MEM      3.     Media Preparation and Sterilization      4.     Contaminations in Animal Tissue Culture

1.     Tissue Collection and Culture      2.     Trypsinization of Cells      3.     Cell Separation by Density Gradient Method      4.     Low Speed Separation of Cells      5.     Isolation of Lymphocyte      6.     Cryo Preservation      7.     Isolation of Spleen Cells      8.     Isolation of Peritoneal Macrophage      9.     Isolation of Blood Dna      10.     Differential Count of Leucocytes      11.     Slide Gel Electrophoresis      12.     Haemagglutination Assay      13.     Elisa      14.     Western Blotting      15.     Widal Test      16.     Identification of Y-chromosomes in Cattle using Pcr

1.     Maintaining Embryonic Stem (ES) Cells      2.     Establishing ES Cells      3.     Freezing of ES Cells      4.     Thawing of Frozen ES Cells      5.     Production of Transgenic using ES Cells      6.     MTT Assay for Cell Viability

1.     Isolation and Purification of DNA from E.coli      2.     Isolation of Plasmid DNA      3.     Gel Electrophoresis of DNA      4.     Southern Blotting      5.     Restriction Digestion

APPENDICES Appendix A   Media Composition      Appendix B   Buffer Composition      Appendix C   Acids Dilutions      Appendix D   Chemical Preparations      Appendix E   Spray Reagents      Appendix F   Acids and Bases References Index