Group Members: Group No.

3
1. Bitaizar, Shaina Nicole Section: NE
2. Hila-os, Kharelle August Ann
Date Submitted:
3. Mangubat, Joah Kim
November 11, 2021
4. Varga, Elainne Marel
5. Yuag, Fer Gus Gere

LAB REPORT Member 1 Member 2 Member 3 Member 4 Member 5

SCORE
Pre-lab Data
Data and Observations
Post-lab Questions
Sub-total
Participation
Online Pre-lab Quiz 6/10 7/10 8/10 10/10 10/10
Total Score

I. Objectives
● To know the process of isolating RNA from yeast and conducting acid hydrolysis with it
● To conduct qualitative tests on both hydrolyzed and unhydrolyzed RNA and observe results
● To observe the given data and use scientific knowledge in answering the questions
● To determine the rationale of the experiment and its scientific function
● To observe the errors and limitations of the experiment and determine their causes and effects

II. Apparatus: III. Symbols of Chemicals:

● Beakers ● Chemical Reagents:
● Stirring Rods ○ Sodium hydroxide - NaOH
● Droppers ○ Distilled water - H2O
● Cheese Cloth ○ Dry yeast - C19H14O2
● Graduated Cylinders ○ Glacial acetic acid - CH₃COOH
● Test Tubes ○ Hydrochloric acid - HCl
● Test Tube Rack ○ Ethanol - C2H5OH
● Litmus Paper ○ Diethyl ether - (C2H5)2O)
● Hot Plate ○ Sulfuric acid - H2SO4
● Centrifuge ○ Silver nitrate - AgNO3
○ Nitric acid - HNO₃
○ Ammonium molybdate - (NH4)2MoO4
○ Sodium carbonate - Na₂CO₃
○ Benedict’s Reagent - C7H10CuNa2O15S
○ Orcinol Reagent - C7H8O2
○ Ammonium hydroxide - NH₄OH

IV. Procedure: (Note: Write schematic diagram on the sheet provided.)

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 V. Data and Observations
ISOLATION OF YEAST RNA
Description of RNA isolated : The isolated RNA appeared as a white, dense substance,
settling at the bottom of the tube, having a thick consistency.

Acid Hydrolysis of RNA

Condition Results

RNA from yeast + 10 mL 10%
H2SO4, covered with marble,
placed in a boiling water bath
for 1 hour + H2O to maintain
the original volume
Upon observing the Acid Hydrolysis of RNA, the
product result which is the hydrolyzed RNA became
a translucent liquid depicting a whitish color. The
volume greatly increased with the addition of the
10 mL sulfuric acid. Also, the visibility of the
hydrolyzed RNA improved upon sitting in a boiling
water bath for about an hour as compared to when
it was shown prior to the boiling process. It was
also observed that the hydrolyzed and the
unhydrolyzed RNA differs in their color as a result
of a different solution added to them.

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 Qualitative Tests
TESTS
Benedict’s Test
2 drops of sample + 4 drops of
Benedict’s rgt. & heated in a boiling
water bath
Orcinol Test
Sample + 4 drops Orcinol reagent
& then heated in a boiling water
bath.
Test for purine bases
5 drops sample + 6M H4OH
until alkaline + few drops 5% AgNO3
Test for inorganic phosphate
5 drops sample + NH4OH in excess
then acidified with 6M HNO3, add 5
drops
(NH4)2MoO4. Warmed and allowed
to stand undisturbed
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OBSERVATIONS
Hydrolysate Unhydrolyzed RNA
The solution yielded a The solution yielded a
reddish-brown color, blue color, signifying an
signifying a presence of absence of reducing
reducing sugar and a sugar and a negative
positive test result. test result.
The solution yielded a The solution yielded an
blueish color, signifying a olive-green color,
presence of pentoses and signifying an absence of
a positive test result. pentoses and a negative
test result.
The solution yielded a The solution yielded a
cloudy/creamy white grayish-white color and
color and filled up to ⅓ of only filled ¼ of the test
the test tube. Also, the tube. Also, the
hydrolysate took more hydrolysate took fewer
drops of 6M NH₄OH for it drops of 6M NH₄OH for
to become alkaline. it to become alkaline.
The solution yielded an The solution yielded a
egg white-like color with murky white color with
an olive green or an olive green or
greyish-green precipitate greyish-green
at the bottom of the tube. precipitate at the
bottom of the tube.
 POST-LAB QUESTIONS

1. How do yeast cells grow and reproduce? (3 pts)

Budding is the process by which yeasts reproduce asexually. As the nucleus divides, a bud forms
on the parent cell’s outer surface. In the elongating bud, one nucleus migrates. Between the bud
and the parent cell, cell wall material forms, and the bud separates. Moreover, yeasts can also
reproduce sexually by forming ascospores, which are formed by fusing the nuclei of two cells and
then going through meiosis. Although sexual reproduction is less common than asexual
reproduction, it allows for genetic recombination.

2. What types of RNA are found in yeast cells? (6 pts)

According to Feldmann (2012), rRNA (80%), mRNA (5% cystolic, ER, mitochondria), tRNAs,
snRNAs, and snoRNAs are among the classes of macromolecules present in Saccharomyces
cerevisiae, a prevalent yeast species. To explain, rRNA molecules are created in the nucleolus, a
specialized part of the cell nucleus that can be divided into large and small subunits, and these
rRNAs interact with ribosomal proteins to form the large and small subunits of the ribosome
(Britannica, 2020). Furthermore, according to Bernstein & Toth (2012), the nuclear exosome in
Saccharomyces cerevisiae is made up of six RNase PH homologues (Rrp41p, Rrp42p, Rrp43p,
Rrp45p, Rrp46p, and Mtr3p). Three possible RNAs Inactive binding proteins (Rrp4p, Rrp40p, and
Csl4p), as well as active exonucleases Rrp44p and Rrp6p, form a scaffolding ring structure.

3. What are the functions of these different RNAs? (6 pts)

As mentioned in number 2, there are different types of RNA found in yeast cells, and these are the
following: rRNA, mRNA, tRNA, snRNA, and snoRNA. And now, these different types of RNA actually
have different functions, which only correspond to each type of RNA.

First, rRNA. rRNA interacts with tRNAs and other molecules that are crucial to protein synthesis.
Hence, rRNAs combine with proteins and enzymes in the cytoplasm to form ribosomes, a
nucleoprotein complex that acts as the site of protein synthesis. Second, mRNA. mRNA carries
complementary genetic code copied from DNA during transcription. Besides, it is responsible for
transporting information to the ribosome. Third, tRNA. tRNA's main function is to transfer amino
acids, which correspond to the mRNA sequence at the ribosome during protein synthesis. Hence, It
also acts as an adapter in the translation of the genetic sequence of mRNA into proteins. Fourth,
snRNA. snRNA's role is to take part in the processing of pre-messenger RNA into mature mRNA.
And Fifth, snoRNA is responsible for sequence-specific nucleotide modification roles. Hence, it
takes part in mRNA editing, modifying rRNA and tRNA, and genome imprinting.

4. What is the purpose of using 1% NaOH in RNA hydrolysis? (5 pts)

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The purpose of 1% NaOH in RNA hydrolysis is crucial to the experiment. It is used to hydrolyze
and make sure that the RNA used in the experiment is not contaminated. Adding strong bases
such as NaOH, will dramatically increase the pH which will decrease the hydrogen ion
concentration of the solution, and denaturing the double-stranded DNA. Moreover, NaOH is a
strong base that lowers the hydrogen ion concentration while raising the pH of the solution. As a
result, after acid extraction at pH 4-5, the nucleic acid will be able to separate from the related
protein and other interfering molecules. Finally, the proper isolation of RNA from yeast in the
experiment will be aided by this.

5. RNA can be hydrolyzed by dilute alkali but not DNA. Why? (5 pts)

RNA can be hydrolyzed by dilute alkali due to their ribose sugar containing a hydroxyl group at
the 2’ position. With this being present, it makes RNA more chemically unstable than DNA since
DNA does not have the 2’ -OH group. This makes DNA not susceptible or vulnerable to
based-catalyzed hydrolysis when being diluted by alkali. Furthermore, unlike RNA, DNA does not
have a hydroxyl group at the 2' position of each sugar group. DNA is significantly more stable in
an alkaline solution as a result of this difference. The hydroxyl group on RNA's 2' position can
release a hydrogen ion into the solution at high pH, generating a highly reactive alkoxide ion that
attacks the phosphate group connecting two neighboring nucleotides (Brennan, 2021).

6. How can you distinguish purines from pyrimidines via hydrolysis procedures? (5 pts)

Purines are larger than pyrimidines because purines have two rings, whereas pyrimidines only
have one. Purines and pyrimidines are nitrogen bases that form hydrogen bonds between DNA
strands. Adenine and guanine are the purines in DNA, just as they are in RNA. Cytosine and
thymine are the pyrimidines in DNA; cytosine and uracil are pyrimidines in RNA. Moreover, the
color, content, and properties of the liquid will be observed as a result of using the hydrolysis
procedure to distinguish purines from pyrimidines. Purine bases are released when ammonium
hydroxide hydrolyzes the n-glycosidic linkage between purine bases and ribose or deoxyribose.
Hence, a silver ion precipitate will produce bubbly viscous material, indicating purine presence
but when such a result isn’t seen, we can deduce that pyrimidine is developed during the
hydrolysis process.

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 For the exclusive use of Chem 153L class in Xavier-Ateneo EXPERIMENT 6

7. Enumerate and draw the structure of the sugar component, purine and pyrimidine
bases present in DNA and RNA. (10 pts)

DNA RNA

A. Sugar Component A. Sugar Component

1. Deoxyribose 1. Ribose

B. Purine Bases B. Purine Bases

1. Adenine (A) 1. Adenine (A)

2. Guanine (G) 2. Guanine (G)

C. Pyrimidine Bases C. Pyrimidine Bases

1. Cytosine (C) 1. Cytosine (C)

2. Thymine (T) 2. Uracil (U)

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 Schematic Diagram of Procedure

I. Isolation of Yeast RNA

Beaker
Dilute 5.0 mL of 1% NaOH Sol’n w/ water
Add 5.0 g Dry Yeast
Heat w/ water bath (15 mins)(stir occasionally)
Beaker + strain the suspension w/ cheesecloth
Transfer the filtrate to two test tubes
Allow to cool down
Centrifuge the filtrate (5-10 mins)
Beaker + the supernatant liquid
Add Glacial Acetic Acid
(until the supernatant is faintly acidic to litmus paper)
Allow mixture to cool (40ºC or lower)
Acidify 10 mL 95% Ethanol by adding 0.2 mL conc. HCl
Cooled mixture + 10 mL Acidified Ethanol
Stir vigorously
Two/four test tubes + (dividing) the mixture into two/four portions
Centrifuge the mixture
Decant the supernatant liquid
Wash the residue/precipitate w/ 2mL 95% Ethanol
Mix well
Combine the mixture into two portions in two test tubes
(if it is divided into four test tubes earlier)
Centrifuge the mixture
Decant the supernatant liquid
Wash the residue w/ 2 mL Ether
Centrifuge the mixture
Decant the supernatant liquid

II. Acid Hydrolysis of RNA

Hydrolyzed RNA Unhydrolyzed RNA

One potion of Isolated RNA (test tube A) One potion of Isolated RNA (test tube B)
+ 10 mL of 10% H2SO4 Dissolve residue w/ 0.05% NaOH
Cover w/ marble (optional) Record observation
Heat w/ boiling water bath (1 hour)
Add water in boiling water bath
(to maintain original volume)
Record observation
III. Qualitative Tests

A. Benedict’s Test

Hydrolyzed RNA Unhydrolyzed RNA

Neutralize 5 drops of Hydrolysate w/ Solid Na2CO3
Decant
Test tube + 2 drops neutralized sample
Add 4 drops Benedict’s Reagent
Heat w/ boiling water bath
Record observation
Test tube + 2 drops Unhydrolyzed RNA
Add 4 drops Benedict’s Reagent
Heat w/ boiling water bath
Record observation

B. Orcinol Test

Hydrolyzed RNA Unhydrolyzed RNA

Test tube + 2 drops of Hydrolyzed RNA
Add 1 drop of conc. HCl
Add 4 drops of Orcinol Reagent
Heat w/ boiling water bath (3 min)
(until greenish-blue solution forms)
Record observation
Test tube + 2 drops Unhydrolyzed RNA
Add 1 drop of conc. HCl
Add 4 drops of Orcinol Reagent
Heat w/ boiling water bath (3 min)
(until greenish-blue solution forms)
Record observation

C. Test for Purine Bases

Hydrolyzed RNA Unhydrolyzed RNA

Test tube + 5 drops of Hydrolyzed RNA Test tube + 5 drops Unhydrolyzed RNA
Add 6M NH4OH (until alkaline) Add 6M NH4OH (until alkaline)
Add few drops 5% AgNO3 Add few drops 5% AgNO3
Record observation Record observation

D. Test for Inorganic Phosphate

Hydrolyzed RNA Unhydrolyzed RNA

Test tube + 5 drops of Hydrolyzed RNA Test tube + 5 drops Unhydrolyzed RNA
Add excess NH4OH Add excess NH4OH
Acidify w/ 6M HNO3 Acidify w/ 6M HNO3
Add 5 drops of Ammonium Molybdate Add 5 drops of Ammonium Molybdate
Reagent Reagent
Warm/ heat w/ water bath (10 mins) Warm/ heat w/ water bath (10 mins)
Record observation Record observation
References:
Bernstein, J., & Toth, E. A. (n.d.). Yeast Nuclear RNA Processing. NCBI. Retrieved November 11, 2021,
from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3272586/
Brennan, J. (2021, November 8). What are the effects of an alkaline pH on the structure of DNA?.
sciencing.com. Retrieved from
https://sciencing.com/effects-alkaline-ph-structure-dna-12030337.html
Britannica, T. Editors of Encyclopaedia (2020, May 22). Ribosomal RNA. Encyclopedia Britannica.
https://www.britannica.com/science/ribosomal-RNA
Feldman, H. (2012). Yeast: Molecular and cell biology (2nd ed). Wiley-VCH Verlag GmbH & Co. KGaA.
10.1002/9783527659180
Kaiser, G. (2021, January 4). Yeasts. Retrieved from Biology LibreTexts:
https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Kaiser)/Unit_4%
3A_Euk
What is the Difference Between Purines and Pyrimidines? (2020, June 1). Retrieved from Albert:
https://www.albert.io/blog/what-is-the-difference-between-purines-and-pyrimidines/

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