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Extraction Of Dna From Calf Or Hog Thymus/Isolation Of Yeast Rna

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Autor:   •  January 4, 2011  •  3,228 Words (13 Pages)  •  1,197 Views

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Extraction of DNA from Calf or Hog Thymus/Isolation of Yeast RNA

I. Abstract

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 cellÐŽ¦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 bakerÐŽ¦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.

II. Results and Discussion

A. Extraction of DNA

Mass of hog thymus = 8.58g

Mass of crude DNA fibers: 0.28g

A260= 0.1102

A280= 0.694

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 ÐŽV 11.038

Table 1 ÐŽV Calibration Plot

A260/A280 % Nucleic Acid

0.57 0.00

0.61 0.25

0.66 0.50

0.71 0.75

0.74 1.00

0.77 1.25

0.80 1.50

0.86 2.00

0.92 2.50

0.97 3.00

1.02 3.50

1.06 4.00

1.15 5.00

1.18 5.50

1.22 6.00

1.25 6.50

1.28 7.00

1.30 7.50

1.33 8.00

1.37 9.00

1.41 10.00

1.49 12.00

1.56 14.00

1.61 17.00

1.67 20.00

By plugging the computed A260/A280 ratios into the trendline equation, the % purity of the DNA extract can then obtained.

y = 15.402x ÐŽV 11.038

% nucleic acid = 15.402 (A260/A280) ÐŽV 11.038

= 15.402 (0.16) ÐŽV 11.038

= -8.57% purity

In calculating for the concentration of the DNA extract, it was assumed that a DNA solution with a concentration of 50 Ñ"Эg/mL has an A260/A280 ratio of 1. The principle of ratio and proportion was then used.

1.00 = 0.16

50 Ñ"Эg/mL DNA concentration

DNA concentration = (0.16)(50 Ñ"Эg/mL)

= 8.0 Ñ"Эg/mL

B. Isolation of RNA

Mass of dry yeast: 3.0 g

Mass of RNA extract: 0.16 g

% recovery: 5.33%

Description of Product: yellowish

% recovery = mass of RNA extract, g x 100

mass of dry yeast, g

= 0.16 g x 100

3.00 g

= 5.33%

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 precaution reduces the chance of contamination from unwanted lipids and proteins that may be present. Cell lysis through fine mincing and homogenizing the hog thymus is accompanied by the addition of citrate buffer---done in order to keep the acidity at near-physiological pH---approx. 7.4 (at this pH, stability of crucial hydrogen bonds and other linkages in the DNA double helix are maintained). The addition of sodium citrate buffer has two other significances. First, it chelates Mg2+ and

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