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BIOCHEMISTRY LABORATORY MANUAL

EXPERIMENT NO. 1
Isolation and Analysis of DNA from Strawberry

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 Strawberries

2. Analyze the isolated DNA using Dische reaction
3. Estimate the purity of the isolated DNA sample by spectroscopic methods

Department of Biochemistry, College Allied Sciences, De La Salle Medical and Health Sciences Institute, 2023
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BIOCHEMISTRY LABORATORY MANUAL

PROCEDURE (Strawberry)

1. Pull off any green leaves on the strawberries. Record its weight.
2. Put the strawberries into a plastic bag (preferrable zip lock bags), seal and gently smash
for about two minutes.
3. In a separate beaker, place homogenizing solution and place approximately 30 – 50 mL
into the plastic bag. Reseal the bag and gently smash it for another minute. Avoid
making too many soap bubble while smashing.
4. Place a cheesecloth on top of a beaker.
5. Open the plastic bag and gently pout he contents into the cheesecloth. You can twist the
cloth above the liquid and gently squeeze the remaining liquid into the beaker.
6. 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.
7. 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.
8. Place the isolated DNA on a pre-weighed microcentrifuge tube or test tube and label.
9. Store the DNA sample in the refrigerator for the next experiment.

Department of Biochemistry, College Allied Sciences, De La Salle Medical and Health Sciences Institute, 2023
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BIOCHEMISTRY LABORATORY MANUAL

DAY 1: ANALYSIS OF DNA FROM STRAWBERRY

MATERIALS

The students need to bring the following:

Aluminum foil cheesecloth

Ziplock bag/plastic bag paperclip
Strawberries (2 pcs per group)
Reagents

Day 1
Homogenizing solution ice cold ethanol
Ice

Day 2
6 M sulfuric acid Dische reagent
Distilled water

Materials

(1) Filter paper (2) 250-mL beaker (1) stirring rod

(1) graduated cylinder (1) trough (1) Watch Glass
(1) Water bath

Equipment

UV-Vis spectrophotometer
Stove

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.

Department of Biochemistry, College Allied Sciences, De La Salle Medical and Health Sciences Institute, 2023
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BIOCHEMISTRY LABORATORY MANUAL

PROCEDURE

A. Calculation of Percent Yield of Isolated DNA

1. Weigh the dried isolated DNA from Day 1.

2. Calculate its percent yield.

B.1 Hydrolysis and Analysis of DNA using Dische Reaction

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

B.2 Dische Reaction

1. Obtain 3 test tubes and number them 1-3.

2. Suspend small amounts of spooled DNA in 3mL distilled water and mix using vortex
mixer.
3. Add to test tube:
a. 2-mL DNA solution (test tube 1)
b. 1-mL DNA solution and 1-mL of Distilled water. (test tube 2)
c. 2-mL of distilled water (test tube 3)
4. Add 2-mL of Dische Reagent to each test tube and mix thoroughly.
5. Place in a boiling water bath for 10 minutes
6. Evaluate your results.
a. A clear tube indicates no nucleic acids.
b. A blue color indicates the presence of DNA.
c. A greenish color indicates the presence of RNA.

C. 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.

Department of Biochemistry, College Allied Sciences, De La Salle Medical and Health Sciences Institute, 2023
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BIOCHEMISTRY LABORATORY MANUAL

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.

PRELAB QUESTIONS (DAY 1) – EXPERIMENT NO. 1

Isolation and Analysis of DNA from Strawberry
Group no. Course/Year-Section

Name Signature

1. ________________________ _______

2. ________________________ _______
Group Members
3. ________________________ _______

4. ________________________ _______

Instructors

1. What are the components of a nucleotide?

2. Consider the structure of a dinucleotide below. Label the following:

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

Department of Biochemistry, College Allied Sciences, De La Salle Medical and Health Sciences Institute, 2023
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BIOCHEMISTRY LABORATORY MANUAL

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.

PRELAB QUESTIONS (DAY 2) – EXPERIMENT NO. 2

Isolation and Analysis of DNA from Strawberry
Group no. Course/Year-Section

Name Signature

1. ________________________ _______

2. ________________________ _______
Group Members
3. ________________________ _______

4. ________________________ _______

Instructors

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

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

Department of Biochemistry, College Allied Sciences, De La Salle Medical and Health Sciences Institute, 2023
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BIOCHEMISTRY LABORATORY MANUAL

3. Which component of DNA will result in a positive reaction to the Dische test?

DATA REPORT SHEET (DAY 1) – EXPERIMENT NO. 2

Isolation and Analysis of DNA from White Onion and Strawberry

Group no. Course/Year-Section

Name Signature

1. ________________________ _______

2. ________________________ _______
Group Members
3. ________________________ _______

4. ________________________ _______

Instructors

A. Isolation of DNA from Strawberry

Mass of strawberries g

Observation after smashing for two minutes.

Observation after putting homogenizing solutions.

Department of Biochemistry, College Allied Sciences, De La Salle Medical and Health Sciences Institute, 2023
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BIOCHEMISTRY LABORATORY MANUAL

Observation after filtration

Estimated volume of the filtrate mL

Estimated volume of ethanol mL

Observation after 3 minutes

DATA REPORT SHEET (DAY 2) – EXPERIMENT NO. 2

Isolation and Analysis of DNA from Strawberry

Group no. Course/Year-Section

Name Signature

1. ________________________ _______

2. ________________________ _______
Group Members
3. ________________________ _______

4. ________________________ _______

Instructors

From Day 1:

Mass of isolated DNA (from Day 1) _________________ g

Percent DNA yield: _______ %

Show your calculations.

Department of Biochemistry, College Allied Sciences, De La Salle Medical and Health Sciences Institute, 2023
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BIOCHEMISTRY LABORATORY MANUAL

A. Hydrolysis and Analysis of DNA using Dische Reaction

Observation
Test
DNA Sample Blank
Hydrolysis
Dische Reaction

B. Dische Reaction without hydrolysis

Test Tube 1 Test Tube 2 Test Tube 3

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

Absorbance at 260 nm _______________

Absorbance at 280 nm _______________

Ratio of A260/A280:

Department of Biochemistry, College Allied Sciences, De La Salle Medical and Health Sciences Institute, 2023
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BIOCHEMISTRY LABORATORY MANUAL

POST-LAB QUESTIONS – EXPERIMENT NO. 2

Isolation and Analysis of DNA from White Onion and Strawberry

GROUP NO.: ________

NAME: _____________________________ CLASS NO. _______

1. What is the purpose of adding homogenizing solution to the sample?

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

3. Comment on the purity of your DNA sample.

Department of Biochemistry, College Allied Sciences, De La Salle Medical and Health Sciences Institute, 2023