This lab aimed to display a few of the fundamental properties that govern carbohydrates. It was demonstrated how differences in molecular weight allow for the separation glucose monomers from starch polymers using a variety of separation techniques. Colorimetric assays were used to test for the presence of the glucose and starch in the fractions and for the analysis of these carbohydrates. A glucose oxidase assay was used to identify the presence of glucose and an iodine assay was used to identify the presence of starch. Valuable hands on skill was also gained from this experiment including use of dialysis bags to assess the permeability of a mixture of carbohydrates based on their size and using a gel filtration column resolve a mixture of carbohydrates based on their molecular size and finally to use colorimetric assays to assess the concentrations of carbohydrates
In this experiment, ion exchange chromatography was performed in order to separate a mixture of glycine, lysine, and aspartic acid. This was achieved by taking advantage of the amino acids’ molecular structures and ionic characters. Separation of these amino acids was accomplished by using a gradient of increasing pH that then was eluted in the order of their isoelectric point. A colorimetric assay was then used to test for the presence of the amino acids using Ninhydrin. As well, during this lab an unknown protein was analyzed by using its molecular weight with a technique known as SDS polyacrylamide gel electrophoresis (SDS-PAGE). The unknown protein was then compared with molecular weight markers and the weight of the unknown protein was calculated using a line of regression equation.
The purpose of experiment 1 was to determine the optimum concentration of enzymes to be used in fixed assays that give you the most constant rate of reaction over 10 mins. The purpose of experiment 2 was to determine the kinetic parameters (KM and Vmax) for the enzyme acid phosphatase using the Hanes plot. The purpose of experiment 3 was to determine the identity of an inhibitor by comparing the kinetic parameters (KM and Vmax) to that of the controls
Two membrane fractions were prepared from a rat liver tissue: a microsomal enriched fraction and a mitochondrial enriched fraction. The lipids were extracted from the microsomal fraction, separated using 2D thin layer chromatography and were then compared with lipids extracted from the entire tissue. The 2D TLC plate is composed of mildly polar silica gel, which will bind polar compounds more tightly than non-polar ones. The first dimension of the plate was developed in a basic solvent and the second dimension was done in a basic solvent. The purpose of these 2 dimensions was to be able visualize the various polarities of the lipids and how they react on the silica gel in varying pH environments. In the second part of the experiment, the lipid/protein ratio in the mitochondrial fraction was analysed. Brilliant Blue Dye R was used to estimate the lipid concentration of the fraction and an absorbance assay was used to estimate the protein concentration.
This lab aimed to display some of the fundamental molecular principles involved in double stranded DNA and to quantify and characterize the DNA using UV spectrophotometry at different wavelengths and by using a melting curve. The purity and the effects of low and high salt on the DNA were also investigated. A plasmid map was constructed to map the relative position of the HindIII and EcoRI restriction enzyme cut sites on a plasmid following digestion of the plasmid with these two enzymes. This map was made by analysing the migration distances of the resulting fragments by agarose gel electrophoresis
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