LABORATORY I (BIOCHEMISTRY AND ANALYTICAL TECHNIQUES)

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.