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 0.2M Ammonium Molybdate



 10% NH4OH


 2% AgNO3



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
2 layers: Brown
bottom.w/ sand Particle
color. No Bilayer
particles–
NH4OH & green w/
gray and particles settled. significant 1st layer:
subside at
10% black
light ppt settled at the changes. brown;
the 2nd
bottom.
Benedict’s
AgNO3 Gray.
at the Before
bottom.boiling Blue to 2 layers were Gray layer:inlight
gray.
Test bottom. – charcoal light blue formed after color,
brown;
gray. Boiled – with boiling clear bilayer,
sand
Ammonium Whitish Yellow,
3 layers: (-) Same No
particle green color Sand
clear ring
particles
Molybdate gray. translucent;
greenish as
settled. significant
on top, particles
structure
subside atat
white curdling; original changes.
translucent brown subside
the at
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 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
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 GROUP 5
Biuret’s 1 drop of No color No color Cloudy light Bilayer –
Test CuSO4 – change. change blue solution 1st layer:
light blue (clear). w/out strand. clear; 2nd
with white layer: blue
10% strands.
Cloudy Clear Clear w/ The strands ppt; foam
Clear,
NH4OH & when not solution, particles. turns black in height:
strands 0.5
are
10% mixed; small a clear present on
AgNO3 white white solution. the glass of
Benedict’s strands
3 layers: suspended
Shade of Blue to Clear light the
Aquatestblue
Test visible
light blue strands.
blue light blue. blue solution tube.
in color.
when
ring on top, lightened. w/out strand.
mixed.
clear in the
middle,
Ammonium White
light blue No color No color No strand only Clear, no
Molybdate particles
layer at the change. change white change.
visible
bottom.w/ (clear). precipitate
white solid 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.

2 ml 10% NaOH sol’n

1 ml sample sol’n
MIX WELL
Add drops 0.5% CuSO4 sol’n
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).

3ml 10% NH4OH

precipitation of complexes of purines with A
2ml filtrate
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 - DO NOT BOIL!
70̊ C
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

Biuret’s Test 10% NH4OH & Benedict’s Test Ammonium

2%AgNO3 Molybdate

purple flocculent, brick red milky yellow

gelatinous white (orange sol’n w/ yellow
RNA ppt solution) ppt

purple flocculent, brick red clear yellow

(eukaryotes gelatinous white (orange sol’n w/ yellow
DNA have histones - ppt solution) ppt
proteins)
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 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.
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!