Report in Nucleic Acid[1]

GROUP I

Aquino, Keziah Christabel

Bahrami, Mohsen

Bantola, Mae Beth

Buenaventura, Marvin

Burhan, Sabreen

Moshaveri, Mehdi

EXPERIMENT #6 ISOLATION OF RNA FROM YEAST

Objectives

1. To be able to isolate RNA from yeast.

2. To be able to get the percentage by mass of RNA from yeast.

3. To be able to identify products of hydrolysis of RNA.

4. To perform tests for identification of products of RNA hydrolysis.

6.2 Hydrolysis of RNA

Materials:

mortar and pestle 0.2% NaOH

Erlenmeyer flask 10% NaOH

yeast 0.5% CuSO4

cheese cloth

sand




Procedure:

1. Grind 4g of yeast in a mortar and equal amount of sand. Further grind the mixture with 15ml of 0.2% NaOH to a smooth, creamy consistency.

2. Transfer the yeast solution to a 125 Erlenmeyer flask and heat the suspension for 30 minutes in a boiling water bath.

3. Filter the solution through a cheese cloth and add 2ml of 10% NaOH and 10 drops of 0.5% CuSO4.




6.3 Identification Tests

Materials:

0.2% NaOH Benedict’s Reagent

0.5% CuS04 0.2M Ammonium Molybdate

10% NH4OH

2% AgNO3



Procedure:

1. Perform the Biuret test to a small amount of filtrate.

2. Transfer 2ml of the filtrate in a test tube. Add 3ml of 10% NH4OH and add 10 drops of 2% AgNO3.

3. To 1ml of the filtrate, add 3ml of Benedict’s reagent. Mix the contents and place it in a boiling water bath.



Procedure:

4. To 0.5ml of the filtrate, add 1ml of 0.2M ammonium molybdate and warm gently at 609-709C. DO NOT BOIL.

5. Record all results.


TEST GROUP 1 GROUP 2 GROUP 3 GROUP 4 GROUP 5

Biuret’s Test 3 drops of CuSO4 – 2 layers: gray and light gray.

Latte color – 2 layers – sand particles at the bottom.

(-) Same as original color.

Purple color after boiling.

Gray in color, some of the particles subside at the bottom.

10% NH4OH & 10% AgNO3

Army green w/ black ppt at the bottom.

Brown w/ sand particles settled at the bottom.

Particle settled.

No significant changes.

Bilayer – 1st layer: brown; 2nd layer: light brown; sand particles subside at the bottom.

Benedict’s Test

Gray. Before boiling – charcoal gray. Boiled – 3 layers: greenish translucent solution; gray, murky, curdling; sand particles.

Blue to light blue with particle settled.

2 layers were formed after boiling clear green color on top, brown undissolved particles at the bottom.

Gray in color, bilayer, clear ring structure at the surface.

Ammonium Molybdate

Whitish gray.

Yellow, translucent; white curdling; sand.

(-) Same as original color.

No significant changes.

Sand particles subside at the bottom.

Objectives

1. To be able to identify products of hydrolysis of DNA.

2. To perform tests for identification of products of DNA hydrolysis.

EXPERIMENT #7 NUCLEIC ACID - DNA


7.1 Hydrolysis of DNA

Materials:

dishwashing detergent 10% NH4OH

disposable cups 2% AgNO3

drinkable H2O 5g NaCl

1 bottle of ice cold Benedict’s Reagent

ethanol (500ml) 0.2% NaOH

wire loop 10% NaOH

filter paper 0.5% CuSO4

ammonium molybdate


1. Dissolve 5g of NaCl in 50ml of H20; add a squirt of dish washing detergent. Save the solution.

2. Gargle about 25ml of H20 in your mouth for 10 minutes and spit into a disposable cup.

3. Add 2cm of the solution from step 2 to a test tube and add 1ml of solution from step 1.

4. Mix the solution by gently inverting the tube 4x.



5. Slowly add 2ml of ice cold ethanol and watch the 2 solutions mix. Note the appearance of tiny white stands.

6. Hook the strands with a glass hook/ wire loop.

7. Perform the same tests in experiment 6 by using 1ml of this extract.

8. Record all results.




TEST GROUP 1 GROUP 2 GROUP 3 GROUP 4 GROUP 5

Biuret’s Test 1 drop of CuSO4 – light blue with white strands.

No color change.

No color change (clear).

Cloudy light blue solution w/out strand.

Bilayer – 1st layer: clear; 2nd layer: blue ppt; foam height: 0.5

10% NH4OH & 10% AgNO3

Cloudy when not mixed; white strands visible when mixed.

Clear solution, small white suspended strands.

Clear w/ particles.

The strands turns black in a clear solution.

Clear, strands are present on the glass of the test tube.

Benedict’s Test

3 layers: light blue ring on top, clear in the middle, light blue layer at the bottom.

Shade of blue lightened.

Blue to light blue.

Clear light blue solution w/out strand.

Aqua blue in color.

Ammonium Molybdate

White particles visible w/ white solid ppt at the bottom.

No color change.

No color change (clear).

No strand only white precipitate was formed and the solution is semi clear.

Clear, no change.

HYDROLYSIS OF NUCLEIC ACIDS

HYDROLYSIS

- is the breaking of bonds by the addition of water.- cleavage of a bond, such as an anhydride or peptide bond, by the addition of the elements of water, yielding two or more products.

NUCLEASE- is an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids.

ACID HYDROLYSIS- is a chemical process in which acid is used to convert cellulose or starch to sugar.

ALKALINE / BASE HYDROLYSIS

- is a chemical process in which a certain molecule is split into two parts by the addition of a molecule of water. One fragment of the parent molecule gains a hydrogen ion (H+) from the additional water molecule. The other group collects the remaining hydroxyl group (OH−).

HYDROLYSIS OF NUCLEIC ACIDS

IDENTIFICATION TESTS

I. BIURET’S TEST

- is a chemical test used for detecting the presence of peptide bonds. In a positive test, a copper(II) ion is reduced to copper(I), which forms a compound with the nitrogens and carbons of the peptide bonds in an alkaline solution. A violet color indicates the presence of proteins.

1 ml sample sol’n

2 ml 10% NaOH sol’n

Add drops 0.5% CuSO4 sol’n

MIX WELL

I. BIURET’S TEST

Biuret reagent:

Potassium hydroxide (KOH)

Hydrated copper (II) sulfate

Potassium sodium tartrate

Ideal Result:

Light blue (-) No protein or peptides

Violet (+) Protein

Pink Peptides (short chain)


II. 10% NH4OH & 2%AgNO3

- is used to detect the presence of purines by precipitation of Ag+ ions. Hydrolysis of N β glycosidic bonds between purine bases and ribose or deoxyribose results in a release of purine bases (adenine and guanine).


2ml filtrate

3ml 10% NH4OH

10 drops 2% AgNO3

precipitation of complexes of purines with Ag+ions

II. 10% NH4OH & 2%AgNO3

Ideal Result:

(+) white ppt


III. BENEDICT’S TEST

- is used as a test for the presence of reducing sugars. This includes all monosaccharides and the disaccharides, lactose and maltose. Benedict‘s reagent contains blue copper(II) ions (Cu2+) which are reduced to copper(I) (Cu+).


1ml of filtrate

3ml Benedict’s reagent

MIX WELL

Boiling water bath

III. BENEDICT’S TEST

Benedict’s reagent:

Sodium carbonate

Sodium citrate

Copper (II) sulfate

Ideal Result:

(+) green, yellow, orange, red, and then brick red or brown (with high glucose present)

(-) blue


IV. AMMONIUM MOLYBDATE

- is a white, crystalline salt used as an analytic reagent, as a precipitant of phosphoric acid, and in pigments. It is used for testing phosphates in nucleic acids. When ammonium molybdate is dropped upon a specimen, it indicates the presence of phosphorus by a yellow stain or a crust of yellow phospho-ammonium molybdate.


0.5ml filtrate

1ml ammonium molybdate

Warm at 60?-70?C

DO NOT BOIL!

IV. AMMONIUM MOLYBDATE

Ideal Result:

(+) yellow stain


1. What are the reagents used to extract DNA and RNA?

RNA:

0.2% NaOH

10% NaOH

CuSO4

DNA:

5g NaCl

Dishwashing detergent

Ice cold ethanol

POST LABORATORY QUESTIONS

2. Did you have the same results of qualitative test performed in experiment 6? Explain.



Biuret’s Test 10% NH4OH & 2%AgNO3

Benedict’s Test

Ammonium Molybdate

RNA

purple flocculent, gelatinous white ppt

brick red (orange solution)

milky yellow sol’n w/ yellow ppt

DNA

purple (eukaryotes have histones - proteins)

flocculent, gelatinous white ppt

brick red (orange solution)

clear yellow sol’n w/ yellow ppt


3. What tests will specifically detect ribose and 2-deoxy-D-ribose? Discuss the principle.

Orcinol Test

Also called Bial's test, is a chemical test for the presence of those sugars or their derivatives which can form furfural upon heating in acidic medium. Furfural formed from pentoses reacts further with orcinol in the presence of FeCl3 to give off blue-green hue of the solution. As ribose do form furfural with a strong acid it makes RNA positive for this test.


Dische Diphenylamine Test

DNA can be identified chemically with the Dische diphenylamine test. The reaction between the Dische reagent and 2-deoxypentose results in the development of a blue color. The reaction depends on the conversion of the pentose to w-hydroxylaevulinic aldehyde which then reacts with diphenylamine to give a blue colored complex. The intensity of the blue color is proportional to the concentration of DNA. Dische reagent does not react with the ribose sugar in RNA and does not form a blue-colored complex.


4. Can DNA be isolated from beef? Discuss the process briefly.

Yes. Here are the steps:

1. Blend a beef liver for 15 seconds with a cup of water and a pinch of salt.

2. Filter the blended beef and put it in a container.

3. Add 2 tablespoons of liquid detergent and mix. Leave for about 5 to 10 mins.

4. Pour the mixture into test tubes.


5. Add small amount of enzymes or meat tenderizers to the test tubes and stir gently (if you stir too hard you might break up the DNA.

6. Pour rubbing alcohol while test tube is titled down the side so that it forms a layer on top of the mixture. Pour until you have about the same amount of alcohol in the tube as the liver mixture.

7. The DNA will rise into the alcohol layer from the liver layer. Use a wire loop to hook or draw the DNA into the alcohol.

8. The slimy material is the DNA.


THANK YOU!

Documents

Report in Nucleic Acid[1]