Rna Extraction
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Methodology
Materials:
1% NaOH
Glacial acetic acid
95% ethanol
Concentrated HCl
Ether
Bakers yeast
Cheesecloth
Equipments:
Top loading balance
Centrifuge
Pasteur pipettes
Thermometer
Litmus paper
Procedure
1. Weigh 3.0 g dry yeast in a 100 mL beaker and add 5.0 mL 1% NaOH and 25.0 mL distilled water.
2. Heat in boiling water bath for 15 minutes with occasional stirring.
3. Strain the suspension through cheesecloth and collect the filtrate in a beaker.
4. Centrifuge the filtrate at maximum speed for 5-10 minutes.
5. Collect the supernatant into another beaker and discard the residue. Add glacial acetic acid drop wise to the supernatant until slightly acidic to litmus.
6. Centrifuge and filter through cheesecloth. Repeat the centrifugation and filtration three to five times, or until the supernatant is clear.
7. Evaporate the supernatant over boiling water bath (or direct heat from a hot plate) to approximately 5.0 mL.
8. Allow the mixture to cool to 40oC or lower.
9. Measure 10 mL of 95% ethanol in a graduated cylinder. Acidify by adding 0.1 mL concentrated HCl.
10. Add the acidified ethanol into the cooled extract with vigorous stirring.
11. Transfer the mixture into test tubes and store in the refrigerator to allow the RNA to settle until the next laboratory period. (Alternately, test tubes can be placed in an ice bath for at least thirty minutes to allow the RNA to precipitate.)
12. Centrifuge the tubes for 5 minutes at 6000 rpm and decant the supernatant.
13. Wash the residue twice with 1.0 mL aliquot of 95% ethanol and twice with ether. Draw off the washings with Pasteur pipette and discard.
14. Add minimum volume of ether to wash residue and transfer the RNA mixture quantitatively with a Pasteur pipette into an evaporating dish.
15. Air dry the mixture. Weigh and calculate the percent recovery.