Experiment No.

08
Isolation and Chemical Characterization of DNA

Biochemistry Lab, BSN1B Date Submitted: November 15,2019

Group No. 1 Date Performed: November14,2019
Class Schedule: 7:00am- 1:00pm
Group Members: Avergonzado, Alaiza

I. Objectives

1. To extract DNA from chicken liver.

2. To analyse qualitatively the principal constituents of DNA.

II. Introduction

The most characteristic constituents of the nucleus are the nucleic acids, DNA and
RNA. Their function have always been of great interest, because of the presence of DNA in
the Chromosomes, and in the transmission of hereditary characteristics. DNA Isolation is one
of the most basic and essential techniques in the study of DNA. The extraction of DNA from
cells and its purification are of primary importance to field of biotechnology and forensics.
Extraction and purification of DNA are the first steps in the analysis and manipulation of
DNA they allow scientists to detect genetic disorders, produce DNA fingerprints of
individuals, and even create genetically engineered organisms that can produce beneficial
products such as insulin, antibiotics and hormones.

DNA can be extracted from many types of cells. The first step is to lyse or break open
the cell. This can be done by grinding a piece of tissue in a blender. After the cells have
broken open, a salt solution such as NaCl and a detergent solution containing the compound
SDS (Sodium dodecyl Sulfate) is added. These solution break down and emulsify the fat &
proteins that make up a cell membrane. Finally, Ethanol is added because DNA is soluble in
water. The Alcohol causes DNA to precipitate, or settle out of the solution, leaving behind all
the cellular components that aren’t soluble in alcohol. The DNA can be spooled (wound) on a
stirring rod and pulled from the solution at this point.

III. Materials

A. Equipment

Refrigerated Centrifuge Blender

Cheesecloth (2) 100 – mL Beaker
Centrifuge Tubes Stirring Rod
25 mL Graduated Cylinder (5) 10 – mL Test Tube
 Dropping Pipette (1) 400 – mL Beaker
Analytical Balance Thermometer

B. Reagents

Chicken Liver Liquid Detergent

2 M NaCI 60% HCIO4
Diphenylamine 5 M H2SO4
5 M NaCI 95% Ethanol (Ice Cold)
Ice – Cold Distilled Water 1 M NH4M0O4
Concentrated HNO3 10% CuSO4
Saturated NaHSO3 2 M NaOH
5 M Ba (OH)2 Bromine Water
1 M Acetate Buffer

IV. Procedure

A. Extraction of DNA

1. Homogenize the 500 grams of frozen chicken in a blender for 2 minutes.

2. Divide 30 mL liver juice for each group.
3. Add 30 mL of 2 M NaCI and 30 mL of Liquid Detergent and stir gently.
4. Heat the mixture to near boiling for 5 minutes.
5. Filter using cheese cloth or coffee filter.
6. Collect the filtrate and Add 2 mL of fresh pineapple juice.
7. Pour 40 mL Ice Cold pure Ethanol gently along the glass rod.
8. After a few second, white fibers (look like cotton) appear in the ethanol layer.
9. Spool the white precipitate and wash with 10% mL 95% Ethanol.

B. Chemical Characterization of DNA

1. Hydrolysis

a. Mix 1/3 of DNA Isolate with 10 Drops of 60% HCI04

b. Heat the mixture at 100°C for 60 minutes with occasional agitation. (Use a
marble to cover the tubes).
c. Upon cooling, add 1 mL of water, grind with a glass rod to produce a
suspension.
d. Centrifuge the supernatant for 10 minutes to separate the solution from the
black particulate residue.
e. Keep the clear supernatant for chemical characterization.

2. Test for Deoxyribose

a. Add 3.5 of Diphenylamine reagent to 1.5 mL of the hydrolysed DNA

 solution.
b. Heat for exactly 10 minutes in a boiling water bath.
c. Cool immediately and observe results.

3. Test for Phosphate

a. Add 1 mL of 5 M H2S04 to 1 mL of the nucleic acid solution.

b. Heat the mixture over a small flame, shaking frequently until the contents of
the tube become brown.
c. Cool. Add 1 drop of conc. HNO3 and heat until white fumes appear.
d. If the solution does not clear, allow to cool and Add 2 more drops of
concentrated HNO3 and Heat.
e. To the colorless liquid add 1 mL Water, heat for 5 minutes in a boiling
water bath. Cool.
f. Add 1 mL of 1 M Ammonium Moly date Solution.
g. Note the result.

4. Test for Purines

a. Place 1 mL of the solution in a test tube and add 1 mL of 2 M NaOH and 2

mL of Acetate Buffer.
b. Heat in a water bath for 15 minutes.
c. Add 10 drops of 10% CuSO4. A Bluish – Brown Precipitate will be formed.
d. Add 10 drops of saturated NaHSO3.
e. Heat in a water bath for a few minutes. A while or light tan Flocculent
precipitate will be observed.

5. Test for Pyrimidines (Cytosine or Uracil)

a. Treat 1 mL of solution with 1.5 mL Bromine Water. Remove excess

Bromine by boiling in a water bath.
b. Add 5 M Ba (OH) 2 in excess.
c. A purple colour indicates Cytosine or uracil and is due to the purple barium
salt of Dial uric acid. The test is negative for Thymine.

V. Results

Chemical Tests Observation

Test For Deoxyribose Black color

Test For Phosphate Yellow precipitate with yellow color of solution

Test for Purines Precipitate is color brown upper is colorless

Test for pyrimidines Light brown precipitate down upper is

colorless no reaction.
VI. Discussion:

The precipitation of DNA was due to the addition of ice-cold ethanol to the solution. DNA
have an ionic nature which makes it insoluble in an aqueous medium which was made less polar by
addition of an organic solvent. Therefore, a white thread-like precipitate was formed. deoxyribose
and it was same with what happened to the standard solution, which indicated a positive result.
Meanwhile, in the test for purines, the DNA color turned from brown to colourless while the
standard guanine solution turned from purple to brown residue which shows a negative result. In
the test for pyrimidine, both the standards formed a purple precipitate while the DNA formed a
white precipitate in a colourless solution which indicates a negative result. The onion DNA showed a
positive result in the test for deoxyribose because the sugar found in the DNA is D-deoxyribose
which reacts with the diphenylamine reagent. However, the visual result should be a blue colored
solution. On the other hand, the DNA must have a positive result with the test for purines and
pyrimidines because the DNA are composed of four bases which are guanine, thymine, adenine and
cytosine.

VII. Conclusion: DNA reacts in acid hydrolysis and less reactive in basic hydrolysis since it is
negatively charged because of the phosphates surrounding it, which means that its isoelectric pH is
above 7 making it basic. DNA is ionic in nature making it insoluble in less polar aqueous medium. As
a result, DNA precipitated out in the presence of ice-cold ethanol because all the other components
of the mixture stayed in the solution except for DNA. The erroneous color results of the standards in
the chemical characterization was because of the reagents used. They might be contaminated or
were unused for a very long time. Because theoretically, a blue solution must be seen as a positive
result in dische test, a red residue in the murexide test, and a purple coloration in the wheeler-
johnson test. Thymine cannot be determined through the test for pyrimidines since it has a methyl
group which prevent its interaction with excess of bromine water. Meanwhile, the error in the DNA
was because of the improper execution of the methods by the experimentalists.

VIII. Assessment (Q&A)

1.What sugar is present in DNA?

5-carbon sugars ribose and deoxyribose are important components of nucleotides, and are found
DNA, respectively.

2.what is the purpose of each of the following components in this protocol? Dishwashing liquid, salt,
meat tenderizer (pineapple juice), ethanol.

Dishwashing liquid-removes fats plus protein to extract Dna meat tenderizer source of Dna Salt-
softens phospholipids in membrane Ethanol-separates Dna from other cell components shows Dna.

3.We can’t really see a Dna molecule under the microscope unless it is rightly coiled into a
chromosome. Why can you see the Dna After you put it into the ethanol?

Dna is released from the wheat ram mixed with water and detergent. The released Dna is
precipitated into the alcohol with after cell components.

4.if you were able to spool the Dna you could see that it is stringly and has the consistency of thick
syrup or mucus. Based on what you know about the molecule; why do you think it has this
consistency?
Dna has a double helix and is like a sparling string. It’s string because it’s wrapped around the
histones which mate s up the chromosomes.

XI. REFERENCES

oyer, R. (2009). Biochemistry Laboratory: Modern Theory and Techniques. San Francisco,
USA: Pearson Education, Inc.,.
Bruns, D., Burtis, C., Ashwood, E., & Sawyer, B. (2007). Fundamental of Molecular
Diagnostics. Philadelphia, PA: Saunders Elsevier.
Hecht, S. (1996). Bioorganic Chemistry: Nucleic Acids. New York, NY: Oxford University
Press.

X. Conformer

Prepared by: Avergonzado, Alaiza