Dna Extraction

METHODOLOGY

DNA extraction via homogenization with liquid nitrogen

Two grams of duck embryo muscle were cut into small pieces and placed into a mortar. Liquid nitrogen was added to the sample and the sample was quickly ground to homogenize it.

The sample was then placed in a 50-mL corning tube containing 10 mL of pre-heated at 55oC 0.05 M Tris-HCl buffer pH 8.0. The mixture was incubated in a water bath at the same temperature for one hour. Approximately 1.10 mL of 10% SDS solution was added to the solution. The solution was then incubated again at the same temperature for 30 minutes. The tube was inverted every ten minutes while being incubated.

After incubation, 5 mL of 24:1 chloroform-isoamyl alcohol mixture was added to the solution. Two phases formed at this point. The mixture was separated equally into two 15-mL corning tubes. The tubes were then centrifuged for ten minutes.

The aqueous upper solution was collected and replaced into the 50-mL tube. Addition of the chloroform-isoamyl alcohol mixture, division and centrifugation were repeated.

After centrifugation, the upper layer was collected and placed in a beaker. One milliliter of 5 M NaOH was added as well as 25 mL of 95% ethanol. The mixture was left to stand to allow precipitation.

The precipitate was then collected using a J-tube and was placed in the fume hood to air dry. After drying, the precipitate was placed in a corning tube and enough 0.05 M Tris-EDTA buffer pH 8.0 was added to make a 10% w/v DNA solution.

Forty microliters of the solution was diluted to 5 mL using the Tris-EDTA buffer. The absorbance of the solution was measured at 260 nm and 280 nm with the Tris-EDTA buffer as blank.

DNA extraction via alkaline lysis

Three pieces of snail egg were placed inside a microcentrifuge tube. Four hundred microliters of 0.1 N NaOH solution was added to the tube before mashing the sample using a pipette tip. The tube was sealed with parafilm and then boiled in a 95-100oC water bath for 25 minutes.

After boiling, 200 μL of distilled water was added as well as 600 μL of 24:1 chloroform-isoamyl alcohol mixture. The mixture will separate into two phases. The tube was inverted several times before it was centrifuged at max speed for 10 minutes.

The aqueous upper phase was collected and placed in a new tube. Care is taken so as not to collect any of the substance in the interphase. The organic phase was discarded.

Four hundred fifty microliters of isopropanol was then added to the tube. The tube was inverted several times before it was put in a freezer for 30 minutes.

The tube was centrifuged for 15 minutes at max speed. The isopropanol was removed without disturbing the DNA pellet. The pellet was then washed with 500 μL of ice-cold 70% ethanol and the tube was centrifuged for five minutes at max speed. The ethanol was then removed.

The DNA pellet was dried over a hotplate at 45oC for 15 minutes. The pellet was then dissolved in 150 μL of 0.5 M Tris-EDTA buffer pH 8.0 before it was stored in the freezer for future use.

DNA extraction using a commercial extraction kit

Tissue the size of two grains of rice from the foot of the snail was cut into small pieces and placed

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