Isolation of yeast RNA
The experiment was first started through mixing of 3.0 g dry yeast, 5.0 ml of 1% NaOH and 25.0 ml distilled water in 100 ml beaker. The resulting mixture was heated in a boiling water bath for 15 minutes while being stirred. The suspension was strained using cheesecloth and the obtained filtrate was obtained and collected in the beaker. This was then centrifuged at maximum speed for about 10-15 minutes. Centrifugation, which uses the idea of gravity, break up the cells and leads to the sedimentation of the largest particles present, the nuclei. Increasing the centrifugal speed brings about the sedimentation of first the cytoplasmic large and small granules. RNA was obtained from the cytoplasmic fractions.
After centrifugation, the supernatant was collected while the residue was discarded. Glacial acetic acid was added in the supernatant until it became slightly acidic. The obtained solution was again centrifuged and filtered through the cheesecloth. Centrifugation and filtration was performed many time until the supernatant became clear. The supernatant, was placed in a boiling water bath on which it is evaporated to approximately 5 ml. The 5 ml mixture was cooled to 40oC and 10 ml acidified 95% ethanol was included with vigorous stirring.
The resulting mixture was placed in an ice bath for at least thirty minutes. This was again centrifuged for 5 minutes at 6000 rpm and the supernatant was discarded. The formed residue was washed with 1ml of 95% ethanol and twice with a small amount of water. The washed crude RNA was transferred in an evaporating dish and was quantitatively determined for its weight. This stock RNA was then added with 0.14 M Tris-HCl buffer to form 10 ml of 10%(w/v) stock RNA solution.tio
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