Isolation of RNA
Materials: Mixer, water bath, suction funnel, conical flasks, beakers, measuring cylinders
(100 cm
3
, 25 cm
3
and 10 cm
3
), centrifuge, UV-spectrophotometer, pH-meter.
Chemicals Yeast, 0.25 M NaOH, conc HAc, 1 M NaCl, 95% ethanol.
Hazards
This practical involves minor risks. Use safety goggles and avoid skin contact with the chemicals. Sodium hydroxide and ethanoic acid are corrosive. Ethanoic acid and ethanol is a fire hazard. Ethanol is injurious to health. Neutralized residues of acid and base can be flushed out in the sink with lot of water.
Task than
To isolate RNA from yeast. Yeast is suitable though yeast contains more RNA
DNA. About 97% is rRNA and 3% is mRNA.
Procedure 1. For 2 lab groups:
Put 50 g yeast and 50 cm
3
0,25 M NaOH in the mixer. Work in the fume cup- board. Use safety goggles. Mix the yeast at high speed for 2 minutes.
The sodium hydroxide will denature the proteins and high pH inhibits the enzyme ribonuclease, which can break down RNA.
Divide the mixture into two portions. Each group will treat their portion according to following:
2. Heat the mixture in a boiling water bath while stirring for 15 minutes. The proteins will completely denature.
3. Filtrate the hot solution in a suction funnel, change filter if necessary. The filtration will remove remainders of cells and denatured proteins. DNA and
RNA are water soluble.
4. Cool the filtrate to room temperature. Add concentrated ethanoic acid under stirring until you get a pH = 6.5. This reduces the hydrolysis of the RNA.
5. Transfer the solution to a centrifuge tube and centrifuge at highest speed for 5 minutes. Proteins will settle at the bottom of the tube.
6. Pour the upper part of the solution into a beaker. Add 1.5 cm few minutes. Pour off the clear upper phase. ethanol. Centrifuge one minute. Dry the RNA on a watch glass.
3
of 1M NaCl under stirring. RNA is soluble in polar solvents as water. By adding NaCl the polarity will increase. Pour the solution while stirring into a beaker with 40 cm
3 of cold ethanol. The ethanol is less polar and the RNA will precipitate. Cold ethanol is used to reduce the hydrolysis of the RNA. Let the mixture stand for a
7. Centrifuge the mixture for a few minutes and pour away the clear upper phase. The precipitate consists mainly of RNA. Wash the RNA with 5 cm
3

8. Take little of the RNA and dissolve it in water. Pour it into a photo spectrometer cell and take up an absorbence spectrum between 220 and 300 nm.
RNA has a typical absorption of 260 nm.