Syllabus (BOTANY)

Course Type: MAJ-11

Semester: 7

Course Code: BBOTMAJ11C

Course Title: Analytical Techniques in Plant Sciences

(L-P-Tu): 4-2-0

Credit: 6

Practical/Theory: Combined

Course Objective: 1. To expose the students to different techniques those can be used to study different biological processes. 2. To provide information about the chemical composition of biomass, characterize its properties and also determine the concentration of both org

Learning Outcome: After the completion of the course the students will be able to: 1. Understand instruments, techniques, lab etiquettes and good lab practices necessary for working in a laboratory. 2. Develop skill in different microscopic techniques. 3. Gain knowledge

(Theory, Credit 4)

Unit	Topic	No. of Lectures/Hrs
1	Imaging and related techniques: Principles of microscopy; Light microscopy; Fluorescence microscopy; Confocal microscopy; Use of fluorochromes: (a) Flow cytometry (FACS); (b) Applications of fluorescence microscopy: Chromosome banding, FISH, chromosome painting; Transmission and Scanning electron microscopy – sample preparation for electron microscopy, cryofixation, negative staining, shadow casting, freeze fracture, freeze etching.	16
2.	Cell fractionation: Centrifugation: Differential and density gradient centrifugation, sucrose density gradient, CsCl2gradient, analytical centrifugation, ultracentrifugation, marker enzymes.	6
3	Radiolabelling techniques: Detection and measurement of different types of radioisotopes normally used in biology, incorporation of radioisotopes in biological tissues and cells, molecular imaging of radioactive material, safety guidelines.	8
4	Chromatography: Principle; Paper chromatography; Column chromatography, TLC, GLC, HPLC, Ion-exchange chromatography; Molecular sieve chromatography; Affinity chromatography.	10
5	Biophysical Method: Molecular analysis using UV/visible, fluorescence, circular dichroism, NMR and ESR spectroscopy, Molecular structure determination using X-ray diffraction, NMR, different types of mass spectrometry.	8
6	Characterization of proteins and nucleic acids: Isolation and purification of RNA, DNA (genomic and plasmid) and proteins, different separation methods. Analysis of RNA, DNA and proteins by AGE, PAGE, SDS-PAGE, and two dimensional gel electrophoresis, Isoelectric focusing gels.	12

Practical (Credits -2, 60 Hrs)

1. Study of Blotting techniques: Southern, Northern and Western, DNA fingerprinting, DNA sequencing, PCR through photographs.
2. To separate sugars by thin layer chromatography.
3. To separate amino acids by paper chromatography.
4. Study of different microscopic techniques using photographs/micrographs (freeze fracture, freeze etching, negative staining, positive staining, fluorescence and FISH).
5. Preparation of permanent slides (double staining).

Reading References:

1. Plummer, D.T. (1996). An Introduction to Practical Biochemistry. Tata McGraw-Hill Publishing Co. Ltd. New Delhi. 3rd edition.
2. Ruzin, S.E. (1999). Plant Microtechnique and Microscopy, Oxford University Press, New York.U.S.A.
3. Ausubel, F., Brent, R., Kingston, R. E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K. (1995). Short Protocols in Molecular Biology. John Wiley & Sons. 3rd edition.

4. Upadhyay, Updhyay and Nath (2009) Biophysical Chemistry : Principles and Techniques, Himalaya Publishing House, Mumbai.

5. Glasel, J. and Deutscher, M. B., "Introduction to Biophysical Methods for Protein and Nucleic acid Research", Academic Press, 1995.

6. Analytical Techniques in Plant Sciences, by Dr. Sanjeeb Kumar Nath , Mahaveer publication , 2022.

7. Practical Manual on Plant Molecular Biology and Analytical Techniques, Dr. S. Girija, AkiNik Publications, 2020