Report in Nucleic Acid[1]
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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.
EXPERIMENT #6
ISOLATION OF RNA FROM YEAST
6.2 Hydrolysis of RNA
Materials:
mortar and pestle 0.2% NaOH
Erlenmeyer flask
10% NaOH
yeast
0.5% CuSO4
cheese cloth
sand
EXPERIMENT #6
ISOLATION OF RNA FROM YEAST
EXPERIMENT #6
ISOLATION OF RNA FROM YEAST
6.2 Hydrolysis of RNA
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.
3. Transfer the yeast solution to a 125 Erlenmeyer flask and heat the suspension for 30 minutes in a boiling water bath. 4.
5. Filter the solution through a cheese cloth and add 2ml of 10% NaOH and 10 drops of 0.5% CuSO4.
EXPERIMENT #6
ISOLATION OF RNA FROM YEAST
6.2 Hydrolysis of RNA
EXPERIMENT #6
ISOLATION OF RNA FROM YEAST
6.3 Identification Tests
Materials:
0.2% NaOHBenedict’s Reagent
0.5% CuS04
10% NH4OH
2% AgNO3
0.2M Ammonium Molybdate
EXPERIMENT #6
ISOLATION OF RNA FROM YEAST
6.3 Identification Tests
Procedure:
1. Perform the Biuret test to a small amount of filtrate. 2.
3. Transfer 2ml of the filtrate in a test tube. Add 3ml of 10% NH4OH and add 10 drops of 2% AgNO3. 4.
5. To 1ml of the filtrate, add 3ml of Benedict’s reagent. Mix the contents and place it in a boiling water bath.
EXPERIMENT #6
ISOLATION OF RNA FROM YEAST
6.3 Identification Tests
Procedure:
4. To 0.5ml of the filtrate, add 1ml of 0.2M ammonium molybdate and warm gently at 60-70C. ̊ ̊ DO NOT BOIL.
5. Record all results.
TEST
GROUP GROUP 2 GROUP GROUP 4 GROUP 5 1 3 Biuret’s 3 drops Latte color – 2 (-) Same Purple color Gray in Test of layers – sand as after boiling. color, some CuSO4 – particles at the original of the 10% Army No Bilayer bottom.w/ sand Particle color. particles– 2 layers: Brown st layer: NH4OH & green w/ particles settled. significant 1 subside at gray and nd 10% black ppt settled at the changes. brown; 2 the bottom. light Benedict’s Gray. Before AgNO3 at the bottom.boiling Blue to 2 layers were Gray layer:inlight gray. Test light blue formed after color, bottom. – charcoal brown; gray. Boiled – with boiling clear bilayer, sand Ammonium Whitish Yellow, (-) Same No 3 layers: particle green color Sand clear ring particles Molybdate gray. translucent; as particles greenish settled. significant on top, structure subside atat white curdling; original changes. subside at translucent brown the the surface. bottom. sand. solution; gray, color. undissolved the bottom. murky, particles at curdling; sand the bottom. particles.
EXPERIMENT #7
NUCLEIC ACID - DNA
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
disposable cups
drinkable H2O
10% NH4OH 2% AgNO3
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
EXPERIMENT #7
NUCLEIC ACID - DNA
7.1 Hydrolysis of DNA
1. Dissolve 5g of NaCl in 50ml of H20; add a squirt of dish washing detergent. Save the solution. 2.
3. Gargle about 25ml of H20 in your mouth for 10 minutes and spit into a disposable cup. 4.
5. Add 2cm of the solution from step 2 to a test tube and add 1ml of solution from step 1. 6.
7. Mix the solution by gently inverting the tube 4x.
EXPERIMENT #7
NUCLEIC ACID - DNA
7.1 Hydrolysis of DNA
1.
6. Slowly add 2ml of ice cold ethanol and watch the 2 solutions mix. Note the appearance of tiny white stands. 7.
8. Hook the strands with a glass hook/ wire loop. 9.
10.Perform the same tests in experiment 6 by using 1ml of this extract. 11.
12.Record all results.
EXPERIMENT #7
NUCLEIC ACID - DNA
7.1 Hydrolysis of DNA
TEST
GROUP 1 GROUP 2 GROUP 3 GROUP 4
Biuret’s Test
1 drop of No color No color CuSO4 – change. change light blue (clear). with white strands. 10% Cloudy Clear Clear w/ NH4OH & when not solution, particles. 10% mixed; small AgNO3 white white suspended Benedict’s strands 3 layers: Shade of Blue to visible Test light blue strands. blue light blue. when ring on top, lightened. mixed. clear in the middle, Ammonium White light blue No color No color Molybdate particles layer at the change. change visible (clear). bottom.w/ white solid
GROUP 5
Cloudy light Bilayer – blue solution 1st layer: w/out strand. clear; 2nd layer: blue foam The strands ppt; Clear, turns black in height: strands 0.5 are a clear present on solution. the glass of the testblue Clear light Aqua blue solution tube. in color. w/out strand. No strand only Clear, no white change. precipitate was formed
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.
HYDROLYSIS 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−).
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
IDENTIFICATION TESTS 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)
IDENTIFICATION TESTS 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 precipitation of complexes of purines with A 10 drops 2% AgNO3
IDENTIFICATION TESTS II. 10% NH4OH & 2%AgNO3
Ideal Result: (+) white ppt
IDENTIFICATION TESTS 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+).
3ml Benedict’s reagent Boiling water bath 1ml of filtrate MIX WELL
IDENTIFICATION TESTS 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
IDENTIFICATION TESTS 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.
1ml ammonium molybdate 0.5ml filtrate Warm at 60̊ 70̊ C
DO NOT BOIL!
IDENTIFICATION TESTS IV. AMMONIUM MOLYBDATE
Ideal Result: (+) yellow stain
POST LABORATORY QUESTIONS 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.
POST LABORATORY QUESTIONS
POST LABORATORY QUESTIONS
RNA
DNA
Biuret’s Test
10% NH4OH & Benedict’s Test Ammonium Molybdate 2%AgNO3
purple
flocculent, brick red gelatinous white (orange ppt solution)
milky yellow sol’n w/ yellow ppt
purple flocculent, brick red (eukaryotes gelatinous white (orange have histones - ppt solution) proteins)
clear yellow sol’n w/ yellow ppt
POST LABORATORY QUESTIONS 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.
POST LABORATORY QUESTIONS
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 whydroxylaevulinic 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.
POST LABORATORY QUESTIONS 4. Can DNA be isolated from beef? Discuss the process briefly. 5.
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.
POST LABORATORY QUESTIONS
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!
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