Advanced Methods in Molecular Biology and Biotechnology. A Practical Lab Manual

Table of Contents

1. Introduction to molecular biology techniques2. Agarose gel electrophoresis3. SDS-polyacrylamide gel electrophoresis4. Immunoelectrophoresis5. DNA plasmid isolation using Alkaline Lysis Method from E. coli DH5a cells6. Restriction digestion of Plasmid DNA7. Genomic DNA extraction from plant leaves using CTAB method8. Genomic DNA extraction from silkworm using modified phenol: chloroform method (Suzuki et al., 1972)9. Genomic DNA extraction from Fungal Mycelium using modified Cenis, 1992 method10. Bacterial transformation protocol using CaCl2

11. Transformation of E. coli by electroporation12. Transfection of cells using lipofectamine13. Total RNA isolation14. RNA isolation from plant tissues with high phenolic compounds and polysaccharides15. Total protein extraction with TCA-acetone and 2D gel electrophoresis16. Dansyl-Edman method of peptide sequencing17. ISSR analysis18. Gene cloning/recombinant DNA technology19. Polymerase Chain Reaction (PCR)20. Blotting/ hybridization and its types21. Dot blot analysis22. ELISA (enzyme-linked immunosorbent assay)23. Study of principle of centrifugation

24. Study of principle of chromatography

25. Thin layer chromatography (TLC)

26. Gel-filtration or size-exclusion chromatography

27. Ion-exchange chromatography

28. To perform affinity chromatography

29. To perform paper chromatography

30. Growth of bacterial cultures and preparation of growth curve

31. To study standard operating procedures (SOPs) for various equipment

Authors

Khalid Z. Masoodi Assistant Professor/Junior Scientist at the Division of Plant Biotechnology, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, India. Dr. Khalid Zaffar Masoodi is a young scientist who works as an Assistant Professor in the Division of Plant Biotechnology at the Sher-e Kashmir University of Agricultural Sciences and Technology in Kashmir (India). He holds a Master's degree in Biotechnology and a doctorate in Plant Biotechnology. Dr. Masoodi is the recipient of numerous awards, including the SERB Early Career Research Award 2016, the SBUR Travel Award in the United States of America in 2012, and the Society of Endocrinology Journal Award in 2018 at Glasgow United Kingdoms. Dr. Masoodi has a distinguished academic record, having qualified for the CSIR-UGC net in Life Sciences, the DBT-BET JRF, and the Graduate Aptitude Test in Engineering and Sciences (GATE) while doing his Masters in Biotechnology. Dr. Masoodi has filed Six patent applications in last 2 years, has over 12 years of teaching and research experience, and has published numerous scientific articles in the fields of cancer research, human disease research, and biotechnology research. His laboratory (Transcriptomics Laboratory, often known as K-Lab, after the Klenow fragment) at SKUAST-K is fully equipped with fluorescence imaging, real-time PCR, cell culture, and Western blotting including other state-of-the-art equipment. His research interests span multiple dimensions, including transcriptomics of plant stress biology (biotic/abiotic) and cancer biology. Apart from that, he is extensively involved in the research of new drugs from medicinal plants. Additionally, he is a co-author of Advanced Molecular Biology and Biotechnology Methods (Academic Press, 2020). Sameena Maqbool Lone Division of Vegetable Science, Sher-e- Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar, Srinagar, India. Sameena Maqbool Lone is a PhD scholar in the Division of Vegetable Science, Sher-e-Kashmir University of Agricultural Sciences and Technology, India. She holds a Master's degree in Horticulture from SKUAST-K, Shalimar. At the Master's level, she worked on "Heterosis and Combining Ability Studies in Cherry Tomato" and obtained competence in morphological and molecular characterization, as well as numerous plant tissue culture technologies. In her Phd programme, she is working on the "Morpho-Molecular Characterization and Nutraceutical Profiling of different carrot genotypes (Daucus carota L.)�. She has outstanding academic and extracurricular achievements, for which she received a merit scholarship from SKUAST-K during her Bachelor's and Master's degree programmes. She has contributed to over 23 academic papers, 6 books, and more than 100 new gene sequences in Genbank by 2022. Rovidha Saba Rasool Division of Plant Pathology, Sher-e- Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar, Srinagar, India. Rovidha Saba Rasool did her Bachelors in Agriculture from SKUAST-K, Wadura followed by Masters in Plant Pathology from SKUAST-Kashmir, Shalimar. Currently, she is pursuing her Ph.D from Division of Plant Pathology, SKUAST-Kashmir. She has done commendable work in finding new genes involved in the pathogenesis of shot hole in stone fruits through insertional mutagenesis in Thyrostroma carpophilum. She has been a good student holding an excellent academic and extracurricular record and was awarded Ph.D. merit scholarship by SKUAST-K, in 2018-19. Apart from this, Rovidha has taken part in various national-level sports competitions and has won many national and state-level awards in baseball and handball.