EXPERIMENT #7: Nucleic Acids

I. Objectives
At the end of the experiment, the student should be able to:
a. isolate RNA; and

b. test qualitatively for the presence of inorganic phosphate, purine bases, and reducing sugars.

II. Material

BEAKER CHEESECLOTH

GRADUATED CYLINDER HEATING SET-UP

DROPPER TEST TUBES

WATER BATH IRON STAND CENTRIFUGE

III. MSDS

Chemical Chemical Molecular Melting Boiling Hazards

name formula weight point point
Sodium NaOH 39.997 g/mol 318 deg cel 1,388 deg Cause eye
Hydroxide cel and skin
irritation.
Acetic acid C2H4O2 60.05 g/mol 17 deg cel 118 deg cel Skin, eye
and
respiratory
irritations
95% Ethyl C2H5OH 46.07 g/mol -114 deg 78.37 deg Corrosive,
alcohol cel cel may cause
fire and
certain
burns
when
exposed to
skin
HCl Hydrochloric 36.46 g/mol -85 deg cel -114 deg May cause
acid cel respiratory
disorders
Orcinol C7H8O2 124.13 g/mol 107.5 deg 290 deg cel Serious eye
cel and skin
irritation
 Benedicts C7H10CuNa2O15S 475.73600 g/ N/A 309 deg cel Not
reagent mol @ 760 considered
mmHg hazardous
Copper CuSO4 159.609 g/mol 110 °C 650 deg cel Harmful if
Sulphate swallowed
and may
cause skin
and eye
irritation

IV. Procedure
A. Separation of Ribonucleic acid (RNA) from yeast B. Hydrolysis of RNA

1. Dilute 10mL of 1% NaOH with 50mL water.2. Add 6 grams

1. Place a small portion of RNA
dried yeast and heat in water bath for 30 minutes with
from yeast in a test tube. 2, Add
occasional stirring.3. Filterthrough cheesecloth and centrifuge
about 10mL of 10% sulfuric acid.
for 10 minutes.4. Decant into a beaker and cool the
Cover the test tube loosely and
supernatant liquid.5. Add glacial acetic acid dropwise until
boil in water bath for 30
faintly acidic to litmus paper, if turbid, transfer in a test tube
minutes.3. Use the solution in
and centrifuge for 5 minutes. Decant.6. Evaporate supernatant
number 2 and the unhydrolyzed
liquid, filter if necessary.7. Cool the filtrate below 40°C then
RNA (dissolved in water) for the
pour in 40mL of 95% ethyl alcohol containing 2 drops of
following tests and compare
conc. HCl.8. Allow RNA to settle, decant and wash with 95%
results:
alcohol. Dry at room temperature.

B.

Orcinol test for ribose –Place 1mL dissolved RNA in a test tube and into another test tube with 1mL of
hydrolysate from number 2. Add 5 drops of orcinol reagent and heat in water bath. Cool the tube immediately under the
faucet and compare the results.Test for Inorganic Phosphates –Add excess NH3to 1mL of the test solution. Acidify with
6N HNO3. Add 1mL of Ammonium Molybdate reagent and heat in water bath.Test for Purine bases –Dissolve a pinch
of the prepared RNA in 1mL of water, add to another test tube, 1mL hydrolysate. Then add 1mL of 2N HCl. Mix and
place in a boiling water bath for 20 minutes. Add 1mL 2N NaOH and 2mL of Acetate buffer, mix and heat in water bath.
Add 0.5mL of 10% CuSO4and note formation of a bluish brown precipitate. Add 10 drops of NaHSO3solution and
mix. After a few minutes of heating, a white or light tan flocculent precipitate forms.Benedict’s test –Neutralize 2mL of
the test solution with Na2CO3. Decant. To 1mL ofthe neutralized sample, add 1mL of Benedict’s reagent. Heat in boiling
water bath and note results.