BIO2 LABORATORY (3rd Quarter)

EXERCISE 2
DNA Extraction

OBJECTIVE:
In this laboratory exercise, you will spend 100 minutes to:
a. Extract DNA from plant material.

MATERIALS:
For this laboratory exercise, you must bring:
1 sachet of liquid detergent
1 pc. 500 mL Ziploc bag
1 pc. ripe banana (Musa sp.)

For this laboratory exercise, you will borrow:

(1) 100 mL beaker (1) water bath

(1) 250 mL beaker (1) electric stove
(1) 100 mL graduated cylinder (1) cheesecloth
(2) 10 mL graduated cylinder (1) thermometer
(1) stirring rod (1) rubber band
(1) dropper
(1) glass slide and cover slip

The following materials are provided by the laboratory technician:

ice liquid detergent
salt cold 95% ethanol

INTRODUCTION
In order to fully grasp the concepts of Molecular Genetics, you must first be equipped with the
basic knowledge of molecular biology which deals with the study of structure and
interactions of cellular molecules such as nucleic acids. Nucleic acids are important
macromolecules that are found in all living things including viruses. They store the needed
genetic information that determines the phenotypic traits of an organism. They are two main
classes of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

DNA is a double-stranded molecule that is responsible for the storage and transfer of genetic
information. It consists of the sugar deoxyribose, phosphate group, and one of the four
nitrogenous bases namely: adenine (A), thymine (T), guanine (G), and cytosine (C). The
two strands of a DNA molecule are complementary to another; that is, the sequence of each
strand determines the sequence of the other. An A on one strand means a T on the opposite
strand, and a G on one strand means a C on the other. 1
RNA is a single-stranded molecule that is responsible for translating the genetic information
into proteins. It consists of the sugar ribose, phosphate groups, and one of the four nitrogenous
bases namely: adenine (A), uracil (U), guanine (G), and cytosine (C). In RNA, thymine (T) is
being replaced by uracil (U).

One of the significant breakthroughs in the field of genetics has been the introduction and
application of molecular tools to understand the genetic trait of an organism. One molecular
technique that is routinely used by geneticists and molecular biologists includes the isolation
of DNA from the nucleus of the desired organism. In DNA extraction, they are three general
steps:

1. Lysis - In this process, cells are being completely disrupted to release the nucleic
acids. It can be done by either physical (grinding/crushing), chemical (application of
chemicals such as detergent), enzymatic (application of enzymes such as
Proteinase K to help disrupt cell walls or tough tissues), or combination of the three.
 2. DNA Precipitation - After nuclear membranes have been broken down, other
unwanted molecules need to be removed such as lipids and proteins. In order to
achieve this, DNA precipitation is being performed. The addition of alcohol and salt
allows DNA to aggregate or precipitate in a visible solid form.
3. DNA Purification - Once DNA has been separated from the aqueous solution, it
will need to be purified to ensure that remaining unwanted cellular debris is
removed by rinsing it with alcohol. After being completely purified, it will be
dissolved in water ready for handling and storage.

METHODS
DNA is present in all cells. However, they are tightly packed into the nucleus of eukaryotic
organisms. In order to extract DNA, it must first be freed from cells. DNA is also water soluble,
so it must be precipitated out in order to obtain DNA.

1. First, produce an extraction buffer solution:

a. Mix 5mL of liquid detergent with 1 gram of salt in a 100mL beaker

b. Add 40mL of tap water to the solution.

c. Stir gently using a stirring rod. Do not let the solution form bubbles/foam. If
any foam or bubbles appear, remove them using the medicine dropper.

2. Place a few pieces of fruit in a 1L Ziploc bag. Squeeze out the air and close the bag.

3. Crush the fruit pieces for roughly 5 minutes. Make sure to not tear holes into the bag.

4. While step two is being done, heat a water bath to 60°C. Adjust the temperature of the
water bath by adding tap water while monitoring the temperature using a thermometer.

5. Place the bags with the crushed fruits in the hot water bath for 10-15 minutes.

6. Then, let the bag cool by transferring it to an ice bath for 1 min.

7. Add the extraction buffer solution to the Ziploc bag.

8. Mix the solution but do not allow bubbles to form.

9. Filter the solution through a cheese cloth over a 250mL beaker. Do not squeeze the cheese
cloth. Let it filter through the cheese cloth on its own. Facilitate this process by slightly
agitating the crushed fruit solution using a stirring rod.

10. Use the medicine dropper to transfer around 3-5 mL of crude fruit solution to a test tube.

11. Slightly tip test tube and gently transfer 10mL of cold ethanol to the solution. Do not
allow the solutions to mix.

12. Allow the text tube to sit for 15 minutes. Try to observe if white, stringy precipitate will
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form. This is the DNA of the fruit.

13. Gently spool it with your stirring rod.

14. Observe how DNA behaves in the test tube.