Biocem Experimnt

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  • Topic: DNA, Protein, Chlorophyll
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  • Published : March 23, 2012
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Experiment 1: Quantitative assessment of Some Cellular Constituents

Summary:
Euglena gracilis are unicellular organisms in the Protist Kingdom. They are known to have both plant and animal characteristics. Although, Euglena cells contain a variety of cellular constituents, their cellular constituents should be presented in equal ratio. The objective of this experiment is to determine Euglena’s cellular components in cells and then to establish their cellular constituents by comparing the experimental results to the expected concentrations given on pg 10 of the lab manual.

Concentration of cells in stock culture used for extraction =9.0×105 cells/ml
Total number of cells used for extraction = 4.05 × 107 cells

Color of Band| Suspected Chlorophyll Pigment| Distance Moved from Origin (cm)| Calculations| Rf Value| Yellow| Violaxanthin/Neoxanthin| 0.7| 0.7cm/5.3cm| 0.132| Light Green| Chlorophyll b| 0.9| 0.9cm/5.3cm| 0.170|

Green| Chlorophyll b| 1.1| 1.1cm/5.3cm| 0.208|
Light Yellow| Lutein & Zoaxanthin| 1.6| 1.6cm/5.3cm| 0.302| Light Green| Chlorophyll a| 2| 2.0cm/5.3cm| 0.377|
Yellow| Carotene| 5.1| 5.1cm/5.3cm| 0.962|

Results:
1) Estimation of Cell Number:
The cell concentration of Euglena sample was determined in order to calculate their cellular constituents. 5 ml sample taken from the stock culture was used to calculate the cell number. The initial absorbance of the Euglena sample measured on the Spectrophotometer 2000 at 675 nm was 2.410 (beyond the maximum allowed abs. of 0.6). The 5 ml sample was diluted with 30 ml water. After a dilution factor of 7 was applied, the absorbance of the sample was brought down to 0.547. By finding the absorbance, the cell concentration can be determined via the Euglena turbidity curve on page 14. An absorbance of 0.547 correlated to a cell number of approximately 9.0×105 cells/ml. The total number of cells used for extraction was: (9.0×105 cells/ml) × (45ml used for extraction) = 4.05 × 107 cells. 2) Thin Layer Chromatography:

a) Chlorophyll Concentration Determination:
Conc. (mg/ml) of total chlorophyll in an acetone extract = Abs. at 652nm / 36 × amount of pooled acetone extract Conc. (mg/ml) of total chlorophyll in an acetone extract = (0.253 / 36)15 = 0.105 mg/ml To find the total chlorophyll per cell, the total chlorophyll in acetone extract was multiplied by the dilution factors and then was divided by the total number of cells per ml in the stock sample. (0.105 x 7 mg/ml) / (4.05 × 107 cells/ml) = 1.81 x 10-8 mg chlorophyll/cell b) Carotenoid Concentration Determination:

Because both carotenoids and chlorophyll are found in the acetone solution, certain correction factors must be applied to the spectrophotometry in order to negate the presence of chlorophyll. Even though carotenoids and chlorophyll have their maximum absorption at 480nm, chlorophyll absorbs in the red region of the spectrum while carotenoids do not. By taking that and the molar extinction coefficients into account, the correction factors effectively eliminate the contributions of chlorophyll. Corrected Abs. at 480 = Abs. at 480nm – [0.114 x (Abs. at 663nm)] – [0.638 x (Abs. at 645nm)] Corrected Abs. at 480 = 0.498 – [0.114 x 0.422] – [0.638 x 0.164] Corrected Abs. at 480 = 0.345

Total conc. (mg/ml) of carotenoid in an acetone extract= Corrected Abs. at 480 / 250 × amount of pooled acetone extract Total conc. (mg/ml) of carotenoid in an acetone extract= (0.345 / 250)15 = 2.07 x 10-2 mg/ml (2.07 x 10-2 x 7 mg/ml) / (4.05 × 107 cells/ml) = 3.58 x 10-9 mg carotenoid/cell

4) Analysis of Supernatant F – DNA:
Amino acids, sugars, nucleotides, acid soluble phosphate compounds, ethanol soluble lipids, fatty acids, glycerophosphatides, sphingolipids, steroids, and ether soluble lipids were extracted sequentially from pellets A-D of Euglena cells. Pellet E was left to dry with...
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