Extraction of DNA from Calf or Hog Thymus/Isolation of Yeast RNA
Nucleic acids may be divided into two groups RNA and DNA. DNA contains almost all the genetic information while RNA serves as the bridge between the DNA and proteins.
Study of both DNA and RNA initially involves proper extraction/isolation. The storehouse of eukaryotic DNA is the nucleus (and in the mitochondria), so experimentally, DNA is extracted from tissues that have a high nuclear to cytoplasmic mass ratio, such as the tissues of the thymus gland and spleen. The thymus gland is a particularly good site for DNA extraction because it functions as the primary site for T lymphocyte differentiation. The T lymphocytes it acts upon have a round nucleus that occupies a greater proportion of the cell¡¦s volume (with only a thin layer of cytoplasm surrounding the nucleus). The high lymphocyte content of the thymus gland, makes DNA extraction much more efficient, convenient and productive. In contrast, RNA extraction is done for cells that have a high cytoplasmic to nuclear mass ratio, such as the fungus Saccharomyces cerevisiae (commonly known as baker¡¦s yeast) used in this experiment.
The extraction procedures for both DNA and RNA basically outline the same steps: release of cell contents (through cell membrane lysis), separation of contaminants (lipids, proteins, etc.) from the desired nucleic acid (DNA, RNA), then precipitation of the separated nucleic acids while making sure that the nucleic acids are not digested or damaged. The samples obtained are then further purified and stored for testing and future use.
The weight of the crude DNA fibers was taken. The extracted DNA was analyzed spectrophotometrically at A260 and A280. The A280/A260 ratio was used to calculate and estimate %DNA purity. The % DNA purity is 29. The weight of the RNA isolate was taken, and from that value % recovery was calculated. The % recovery was 5.33.
Results and Discussion
Extraction of DNA
Mass of hog thymus = 8.58g
Mass of crude DNA fibers: 0.28g
A260/A280 = 0.1588 ~ 0.16
A pure DNA solution would have a ratio of 1.8-2.0.
%DNA purity= -8.57%
A calibration plot was constructed, based on the table of values given in page 28 of the Chemistry 40.1 laboratory manual. The inverse of the values on the left column (A280/A260) were calculated to yield the A260/A280 values shown in Table 1 below. The values for % nucleic acid were then plotted against the A260/A280 ratios to come up with Figure 2. A trendline equation was obtained as well:
y = 15.402x ¡V 11.038
Table 1 ¡V Calibration Plot
% Nucleic Acid
By plugging the computed A260/A280 ratios into the trendline equation, the % purity of the DNA extract can then obtained.
15.402x ¡V 11.038
% nucleic acid
15.402 (A260/A280) ¡V 11.038
15.402 (0.16) ¡V 11.038
In calculating for the concentration of the DNA extract, it was assumed that a DNA solution with a concentration of 50 Ýg/mL has an A260/A280 ratio of 1. The principle of ratio and proportion was then used.
Isolation of RNA
Mass of dry yeast: 3.0 g
Mass of RNA extract: 0.16 g
% recovery: 5.33%
Description of Product: yellowish
mass of RNA extract, g
mass of dry yeast, g
The first step in the experiment involving the extraction of DNA from hog thymus is cell lysis. The hog thymus was first thoroughly cleaned by removing residual fat, blood vessels, and other extraneous tissues as this...
References: Horton, Robert H., et al. Principles of Biochemistry. 3rd ed. Prentice-Hall Publishers. New Jersey. 2002.
Biochemistry Laboratory Manual, UP Diliman, 2002
Zubay, Geoffrey L. et al., Principles of Biochemistry, Wm. C. Brown Publishers, Iowa, 1995.
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