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MODERN RESEARCH TECHNIQUES (BIT-319) Credit Hours 3(2-1) Educational Objectives: This particular course is designed to provide students understanding of basic concepts of research and its methodologies. The students will learn about modern techniques used in the field of applied biosciences. This will develop research interest and will help them identify local problems and finding the appropriate solutions using biotechnological procedures. PRE- REQUISITE(s): Thorough knowledge of Microbiology, Vaccinology and Virology is required. MODERN RESEARCH TECHNIQUES (BIT-319) Credit Hours 3(2-1) Educational Objectives: This particular course is designed to provide students understanding of basic concepts of research and its methodologies. The students will learn about modern techniques used in the field of applied biosciences. This will develop research interest and will help them identify local problems and finding the appropriate solutions using biotechnological procedures. PRE- REQUISITE(s): Thorough knowledge of Microbiology, Vaccinology and Virology is required.

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Course Contents 1.DNA and RNA isolation 2.Quantification of DNA and RNA 3.Primer designing 4.PCR 5.Electrophoresis 6.Sequencing 7.Karyotyping 8.Restriction Mapping Course Contents 1.DNA and RNA isolation 2.Quantification of DNA and RNA 3.Primer designing 4.PCR 5.Electrophoresis 6.Sequencing 7.Karyotyping 8.Restriction Mapping 9.Flow cytometry 10.Hybridization a.Western blotting b.Southern blotting c.Northern blotting d.FISH 9.Flow cytometry 10.Hybridization a.Western blotting b.Southern blotting c.Northern blotting d.FISH

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11.Transfection 12.Transduction 13.Transformation 14.Cloning 15.Microarrays 16.Chromatography 17.Immunochemistry 18.ELISA 19.Bioinformatics and techniques 20.Ethical issues 11.Transfection 12.Transduction 13.Transformation 14.Cloning 15.Microarrays 16.Chromatography 17.Immunochemistry 18.ELISA 19.Bioinformatics and techniques 20.Ethical issues 21.Tissue culturing 22.Slide Preparation and Cell Stains 23.Agar plate preparation and streaking for the purpose of individual colony isolation 24.Bacterial Growth on selective agar 25.Quantification: Colony Forming Units (CFU) 26.Dilution Plating 27.Identification and characteristics of colonies 21.Tissue culturing 22.Slide Preparation and Cell Stains 23.Agar plate preparation and streaking for the purpose of individual colony isolation 24.Bacterial Growth on selective agar 25.Quantification: Colony Forming Units (CFU) 26.Dilution Plating 27.Identification and characteristics of colonies

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Lab Work: DNA extraction Quantification PCR Electrophoresis ELISA Agar plate preparation Streaking Transfection Cloning Lab Work: DNA extraction Quantification PCR Electrophoresis ELISA Agar plate preparation Streaking Transfection Cloning

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Recommended Books: Immunoassay and other bioanalytical techniques by Eman.J.Immunoassay Nano biotechnology by C.M. Niemeyer, C.A. Mirkin MLT (medical laboratory technology): methods and interpretation by Rammiksood Genetic techniques for biological research: a case study approach by Corinne V. Anthony MichelsGenetic techniques for biological research: a case study approach Corinne V. Anthony Michels Research techniques in biochemistry and molecular biology by Robert E. Thach, Mary R. NewburgerResearch techniques in biochemistry and molecular biologyRobert E. ThachMary R. Newburger Methods in molecular biology and protein chemistry: cloning and characterization of an enterotoxin subunit by Brenda D. Spangler.Brenda D. Spangler Recommended Books: Immunoassay and other bioanalytical techniques by Eman.J.Immunoassay Nano biotechnology by C.M. Niemeyer, C.A. Mirkin MLT (medical laboratory technology): methods and interpretation by Rammiksood Genetic techniques for biological research: a case study approach by Corinne V. Anthony MichelsGenetic techniques for biological research: a case study approach Corinne V. Anthony Michels Research techniques in biochemistry and molecular biology by Robert E. Thach, Mary R. NewburgerResearch techniques in biochemistry and molecular biologyRobert E. ThachMary R. Newburger Methods in molecular biology and protein chemistry: cloning and characterization of an enterotoxin subunit by Brenda D. Spangler.Brenda D. Spangler

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DNA ISOLATION Why to isolate where to isolate

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1- Cell lysis 1.Sonication, 2.vortexing with pheno(cell walls n capsids) 3.SDS for lipid 4.protease. DNA associated+cellular proteins 2- Protein Precipitation a.salt ammonium or sodium acetate. b.phenol-chloroform: vortexed n entrifuged c.proteins will remain in the organic phase and can be drawn off carefully. MANUAL How to isolate

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DNA precipitation: mixing with cold ethanol or isopropanol and then centrifuging. alcohol serves as a wash to remove the salt previously added. DNA precipitation: mixing with cold ethanol or isopropanol and then centrifuging. alcohol serves as a wash to remove the salt previously added. Washing: the resultant DNA pellet with cold alcohol again and centrifuge for retrieval of the pellet. Washing: the resultant DNA pellet with cold alcohol again and centrifuge for retrieval of the pellet. Drying: the pellet, the DNA can be re-suspended in a buffer such as Tris or TE. Drying: the pellet, the DNA can be re-suspended in a buffer such as Tris or TE. Visualization: Presence of DNA can be confirmed by electrophoresing on an agarose gel containing ethidium bromide, or another fluorescent dye that reacts with the DNA, and checking under UV light Visualization: Presence of DNA can be confirmed by electrophoresing on an agarose gel containing ethidium bromide, or another fluorescent dye that reacts with the DNA, and checking under UV light

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PH 7= DNA +RNA in aqueous PH 4= DNA precipitated PH 7= DNA +RNA in aqueous PH 4= DNA precipitated

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Chelating agent to (EDTA) sequester divalent cations such as, Mg 2+ and Ca 2+, which prevents enzymes like DNase from degrading the DNA.

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unsuitable for purification of short (