1. 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.

2. 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

3. 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

4. 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

5. 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

6. DNA ISOLATION Why to isolate where to isolate

7. 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

8. 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

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

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

12. unsuitable for purification of short (<200 nucleotides) RNA species, such as siRNA, miRNA, gRNA and tRNA.nucleotidessiRNAmiRNAgRNAtRNA SPIN COLUMN BASED NUCLEIC ACID PURIFICATION

13. AUTOMATED DNA PURIFICATION

14. RNA ISOLATION Total RNA Total nucleic acid (RNA + DNA) MicroRNA & small RNA mRNA Sequence-specific RNA Purified RNAs Viral RNA

15. Blood Cultured cells FFPE samples Liquid and cell-free liquid Plant Tissue Yeast Bacteria Exosomes where to isolate Northern blot analysis RNA sequencing RNA microarray analysis Transcriptome analysis RNA cloning Real-time RT-PCR RNA structure/function Ribosomal RNA depletion In vitro transcription & translation Why to isolate

16. HOW TO ISOLATE RNA

17. RNase contamination during RNA extraction. Harder than dnases Cellular Environmental RNase 7, a member of the RNase A superfamily is secreted by human skin equipment used for RNA extraction is usually cleaned thoroughly, kept separate from common lab equipment and treated with various harsh chemicals that destroy RNases. PRECAUTIONS

18. DNA and RNA Quantification In spectroscopy, the absorbance (also called optical density of a material is a logarithmic ratio of the radiation falling upon a material, to the radiation transmitted through a material

19. The ratio of the absorbance at 260 and 280nm (A 260/280 ) is used to assess the purity of nucleic acids. For pure DNA, A 260/280 is ~1.8 and for pure RNA A 260/280 is ~2. The ratio of the absorbance at 260 and 280nm (A 260/280 ) is used to assess the purity of nucleic acids. For pure DNA, A 260/280 is ~1.8 and for pure RNA A 260/280 is ~2. Sample purity At a wavelength of 260 nm, the average extinction coefficient for 1.double-stranded DNA is 0.020 (μg/ml) -1 cm -1, 2.single-stranded DNA it is 0.027 (μg/ml) -1 cm -1, 3.single-stranded RNA it is 0.025 (μg/ml) -1 cm -1 4.short single-stranded oligonucleotides it is dependent on the length and base composition. At a wavelength of 260 nm, the average extinction coefficient for 1.double-stranded DNA is 0.020 (μg/ml) -1 cm -1, 2.single-stranded DNA it is 0.027 (μg/ml) -1 cm -1, 3.single-stranded RNA it is 0.025 (μg/ml) -1 cm -1 4.short single-stranded oligonucleotides it is dependent on the length and base composition. Mass attenuation coefficient measurement of how strongly a chemical specie or substance absorbs or scatters light at a given wavelength, per unit mass. Mass attenuation coefficient measurement of how strongly a chemical specie or substance absorbs or scatters light at a given wavelength, per unit mass.