Pre-Lab Report

Experiment No. 3 – Midterm Period

Day 1 - Isolation of DNA from White Onion
Group No.: ________ CYS: __________ Class No. _______
Name: _____________________________________________________________
Date Submitted: ______________________ Signature: ________________

1. What are the components of a nucleotide?

2. Draw the structure of the dinucleotide AT. Indicate the following:

a. 5’ and 3’ ends
b. the phosphodiester bond
c. N-glycosidic bonds

3. List the composition of the homogenizing solution used in the extraction of DNA from
white onion. Describe the role of each reagent found in this mixture.
EXPERIMENT NO. 3 – Midterm Period
Isolation and Analysis of DNA from White Onion

I. BACKGROUND

Nucleic acids are polymers of nucleotides linked by 3’ to 5’ phosphodiester bond.

Nucleotides are the building blocks of nucleic acids and each one is composed of a nitrogeneous
base, a pentose sugar backbone, and a phosphate group. The nitrogeneous bases can be
grouped into two as either a purine or a pyrimidine. Adenine and guanine are purine bases while
cytosine, thymine, and uracil are pyrimidine bases. Thymine is only found in DNA and it is
replaced by uracil in RNA.

There are two types of nucleic acids, the DNA and the RNA. DNA serves as repository of
genes which carry the biological instruction for the synthesis of proteins while RNA is directly
involved in protein synthesis.

Nucleic acids can be analyzed spectroscopically because of their bases. Purines and
pyrimidines absorb light around 260 nm, while the aromatic amino acids (found in protein) absorb
around 280 nm. By calculating the ratio of the absorbance at 260 nm and at 280 nm (A260/A280),
we can estimate the purity of the isolated sample. A ratio of 1.8 is a characteristic of a pure DNA
sample, while a ratio of 0.6 is characteristic of a pure protein.

II. OBJECTIVES

At the end of this activity, the students should be able to:

1. Isolate DNA from white onion
2. Analyze the isolated DNA using Dische reaction
3. Estimate the purity of the isolated DNA sample by spectroscopic methods
 DAY 1: ISOLATION OF DNA FROM WHITE ONION

III. MATERIALS

The students need to bring the following:

white onion
paper clip, hairpin, or thin wire to be used as a hook
cheesecloth or old handkerchief

Reagents
homogenizing solution ice
ice-cold ethanol (95% or absolute)

Materials
(1) Filter paper
(1) Knife and chopping block
(2) 250-mL beaker
(1) stirring rod
(1) graduated cylinder
(1) trough
(1) thermometer
(1) watch glass

Equipment
Stove
Laboratory blender

IV. SAFETY

• Wear lab gown and protective goggles at all times.

• Place highly volatile samples under fume hood.
• Wash hands thoroughly with soap and water before leaving the laboratory.
PROCEDURE

1. Chop an onion into small pieces. Weigh 20 – 25 g (to the nearest 0.01 g) directly into a
250-mL beaker. Record its weight.
2. Into a separate 250-mL beaker, pour the homogenizing solution. For every 1 gram of
white onion sample, use 2 mL of homogenizing solution. Record the volume of
homogenizing solution used.
3. Heat the homogenizing solution in a warm water bath until the temperature reaches 60°C.
4. When the temperature reaches 60°C, immediately add the chopped onion.
5. Stir and incubate the mixture for 15 minutes. Maintain the 60°C temperature.
6. After the incubation period, place the beaker containing the mixture in an ice bath and
incubate for 5 minutes with mild stirring.
7. Homogenize the mixture using a blender for 45 seconds at low speed.
8. Pour the homogenate (the product of homogenization) into a 250-mL beaker and incubate
it in an ice bath for 15 to 20 minutes.
9. Filter the cooled homogenate through 2 layers of cheesecloth over a 250-mL beaker
placed in an ice bath. Try to estimate the volume of the filtrate based on the reading on
the beaker.
10. Tilt the beaker at a certain angle and slowly add ice-cold 95% or absolute ethanol down
the side of the beaker. The amount (volume) of the ethanol must be twice the amount
of the filtrate.
11. Allow the mixture to stand for 3 minutes. Collect the floating DNA in one direction only,
using a spooler. The spooler can be a clean wire that has a hook shape at one end.
12. Place the isolated DNA on a pre-weighed watch glass and filter paper and allow it to dry
in your locker.
13. Calculate the percent yield.
mass of recovered DNA
%yield = ´ 100
mass of white onion
 DATA REPORT SHEET
EXPERIMENT NO. 3 – Midterm Period
DAY 1 - Isolation of DNA from White Onion

Group No. ______ CYS : _______

Date Performed: __________________ Date Submitted:_________________
Names with signature: (alphabetical, surnames first) Class Nos.
____________________________ ________________ __________
____________________________ ________________ __________
____________________________ ________________ __________
____________________________ ________________ __________

Isolation of Casein from Milk

Mass of white onion g

Observation after 15 minutes of incubation

Observation after 5 minutes of incubation

Observation after homogenization

Observation after 20 minutes of incubation

Estimated volume of the filtrate mL

Estimated volume of ethanol mL

Observation after 3 minutes

Percent DNA yield: Show your calculations.

 Pre-Lab Report
Experiment No. 4 – Midterm Period
Day 2 – Analysis of DNA from White Onion
Group No.: ________ CYS: __________ Class No. _______
Name: _____________________________________________________________
Date Submitted: ______________________ Signature: ________________

1. What is the chemical component of the Dische reagent?

2. What color will indicate a positive reaction to the Dische test?

3. Which component of DNA will result in a positive reaction to the Dische test?
 DAY 2: ANALYSIS OF DNA FROM WHITE ONION
III. MATERIALS

The students need to bring the following:

Isolated DNA from white onion (from Day 1)
Aluminum foil

Reagents
6 M sulfuric acid
Dische reagent
Distilled water

Materials
Test tubes dropper

Equipment
stove
water bath
laboratory blender
UV-Vis spectrophotometer

IV. SAFETY

• Wear lab gown and protective goggles at all times.

• Place highly volatile samples under fume hood.
• Wash hands thoroughly with soap and water before leaving the laboratory.
PROCEDURE

A. Hydrolysis and Analysis of DNA using Dische Reaction

2. Place the isolated DNA into a test tube and add 20 drops of sulfuric acid. Cover with a foil.
3. Heat the test tube in a boiling water bath for one (1) hour with occassional shaking.
4. Cool the mixture at room temperature and add 2.0 mL of water.
5. Place 20 drops of the hydrolyzed DNA sample into a clean test tube.
6. To another test tube, add 2 mL of water. This will serve as your blank.
7. To each tube, add 2.0 mL of Dische reagent
8. Heat the tubes for 10 minutes in a boiling water bath.
9. Cool the solutions and note your observations.

B. Purity of the Isolated DNA by UV-Vis Spectroscopy

1. Dissolve a small amount of your isolated DNA pellet with 3 mL of distilled water.
2. Get 1 mL of your dissolved pellet and place transfer it to a cuvette.
3. Record the absorbance at 260 nm and at 280 nm using the UV-Vis spectrophotometer.
Use distilled water as a blank.

WASTE DISPOSAL: Dispose all wastes in the Organic Waste Bottle.

 DATA REPORT SHEET
EXPERIMENT NO. 4 – Midterm Period
DAY 2 - Analysis of DNA from White Onion

Group No. ______ CYS : _______

Date Performed: __________________ Date Submitted:_________________
Names with signature: (alphabetical, surnames first) Class Nos.
____________________________ ________________ __________
____________________________ ________________ __________
____________________________ ________________ __________
____________________________ ________________ __________

A. Hydrolysis and Analysis of DNA using Dische Reaction

Observation
Test
DNA Sample Blank

Hydrolysis

Dische Reaction

B. Purity of the Isolated DNA by UV-Vis Spectroscopy

Absorbance at 260 nm _______________

Absorbance at 280 nm _______________

Ratio of A260/A280: _______________

 POST-LAB REPORT
Experiment Nos. 3 and 4 – Midterm Period
Isolation and Analysis of DNA from White Onion
Group No.: ________ CYS: __________ Class No. _______
Name: _____________________________________________________________
Date Submitted: ______________________ Signature: ________________

1. Why was the incubation temperature set to 60°C?

2. What was the purpose of incubating the homogenate in an ice bath?

3. What was the purpose of adding ice-cold ethanol?

4. Comment on the purity of your DNA sample.