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LABORATORY I
Course Objectives: This course will impart practical skills to students to perform experiments related to quantitation of biomolecules using analytical techniques, isolate, identify and enumerate microbes and study their metabolic capability, mutant isolation and solve problems based on genetics.
Course Outcomes (COs):
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Course |
Learning outcome (at course level) |
Learning and teaching strategies |
Assessment Strategies |
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Course Code |
Course title |
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24MBL 128 |
Laboratory I (Practical) |
CO 43: Elaborate concepts of biochemistry by performing assays related to estimation of various biomolecules. Estimate the quantity of given protein and illustrate the kinetic parameters of the given enzyme. CO44: Formulate media and design experiments for isolation, identification and enumeration of microbes present in a sample and analyze their metabolic capability of microbes using biochemical tests. CO45: Apply replica plating and gradient plate techniques for isolation of mutants. CO46: Identify and illustrate the various elements of biochemistry, analytical techniques, microbiology and genetics CO47: Discuss and defend the concepts of biochemistry, analytical techniques, microbiology and genetics and illustrate the exercises performed with appropriate methods and outcomes and maintain proper documentation of the same. CO48: Contribute effectively in course-specific interaction
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Approach in teaching: Hands-on practical, demonstrations, simulations
Learning activities for the students: Discussion, Tutorials, Assignments Reading Journals
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Class test, Semester end examinations, Hands-on practical assessment |
Experiments based on:
Biochemistry and Analytical Techniques
1. Preparing various stock solutions and working solutions that will be needed for the course.
2. To prepare an Acetic-Na Acetate Buffer and validate the Henderson-Hasselbach equation.
3. To determine an unknown protein concentration by plotting a standard graph of BSA using UV-Vis Spectrophotometer and validating the Beer- Lambert’s Law.
4. Titration of Amino Acids and separation of aliphatic, aromatic and polar amino acids by thin layer chromatography.
5. Purification and characterization of an enzyme from a recombinant source (such as Alkaline Phosphatase or Lactate Dehydrogenase or any enzyme of the institution’s choice).
a) Preparation of cell-free lysates
b) Ammonium Sulfate precipitation
c) Ion-exchange Chromatography
d) Gel Filtration
e) Affinity Chromatography
f) Dialysis of the purified protein solution against 60% glycerol as a demonstration of storage method
g) Generating a Purification Table (protein concentration, amount of total protein; Computing specific activity of the enzyme preparation at each stage of purification)
h) Assessing purity of samples from each step of purification by SDS-PAGE Gel Electrophoresis
i) Enzyme Kinetic Parameters: Km, Vmax and Kcat.
6. Experimental verification that absorption at OD260 is more for denatured DNA as compared to native double stranded DNA. Reversal of the same following DNA renaturation. Kinetics of DNA renaturation as a function of DNA size.
7. Identification of an unknown sample as DNA, RNA or protein using available laboratory tools.
8. Biophysical methods (Circular Dichroism Spectroscopy, Fluorescence Spectroscopy).
9. Determination of mass of small molecules and fragmentation patterns by Mass Spectrometry.
Microbiology
10. Study of various symptoms produced in plants due to virus infection.
11. Study of viral diseases of plants/animals/human (Specimen/photographs)
12. Different type of viruses (Photographs/sketches).
13. Titration of Phages
14. Isolation and identification of fungi.
15. Study of permanent slides of algae and fungi
16. Isolation and identification of Rhizobium from root nodules of leguminous plants.
17. Isolation and identification of Azotobacter from soil.
18. Sterilization, disinfection and safety in microbiological laboratory.
19. Preparation of media for cultivation of bacteria.
20. Isolation of bacteria in pure culture by streak and spread plate methods.
21. Maintenance of stock cultures: slants, stabs and glycerol stock cultures
22. Study of colony and growth characteristics of some common bacteria: Bacillus, E. coli, Staphylococcus, Streptococcus, etc.
23. Preparation of bacterial smear and Staining methods: Negative, Gram’s staining, endospore staining.
24. Enumeration of bacteria: standard plate count.
25. Biochemical characterization of microbes.
26. Isolation and identification of bacteria from soil/water samples.
27. Determination of phenol co-efficient of antimicrobial agents.
Genetics
28. Isolation of antibiotic resistant microorganisms by replica plating.
29. Isolation of antibiotic resistant microorganisms by Gradient plate technique
30. Simple problems based on Mendel’s laws and gene interactions.
