School of Food Science and Environmental Health Laboratory Report.

Module Code and Title: TFBG1002

Lecturer:
Furong Tian

Experiment Title: DNA Isolation from Onion

Experiment Date: 03/02/2020

Submission Date:
17/02/2020

Student name(s) and number:

Olga Ciudin C19757639

Course Code: DT425 – Year I

Aims and Objectives

The purpose of this experiment is to gain experience into the procedure of DNA
isolation, separating it from other cellular components in a way that still preserves its
structure and sequence from onion cell.
The objective is to demonstrate several basic steps required for isolation of chromosomal
DNA from cells, as well to make observations regarding the results of the isolation and to develop a
hypothesis from taken observations regarding the results of the isolation.

Introduction

The chemical DNA was first discovered in 1869, but its role in genetic inheritance
was not demonstrated until 1943. In 1953 James Watson and Francis Crick, aided by the
work of biophysicists Rosalind Franklin and Maurice Wilkins, determined that the structure
of DNA is a double-helix polymer, a spiral consisting of two DNA strands wound around
each other. The breakthrough led to significant advances in scientists’ understanding of DNA
replication and hereditary control of cellular activities. (Encyclopaedia Britannica, 2020)
Nucleic Acid is one of the important biochemical molecules present in an living
being. It functions for encoding, transmitting, and expressing the genetic information. It has
two types which is the DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). DNA, or
deoxyribonucleic acid, is the hereditary material in humans and almost all other organisms.
(Genetics home reference, 2020)
The ability to extract DNA is of primary importance to studying the genetic causes of
disease and for the development of diagnostics and drugs. It is also essential for carrying out
forensic science, sequencing genomes, detecting microorganisms and viruses in the
environment and for determining paternity. (What is biotechnology, 2020)
Onions are used as the best material for extraction of DNA as they have low starch
level and a large number of DNA, which gives opportunity to observe DNA strands more
clearly. In order to extract DNA from onion cells, chemical properties are used in labs, to
perform this procedure. The three basic steps of DNA extraction are 1) lysis, 2) precipitation,
and 3) purification. (Sirakov, 2016)
To extract the chromosomal DNA, both the cell membrane and the nuclear membrane
must be lysed, or broken open. This is accomplished by disrupting the membranes with a
solution of detergent and salt, creating a cell homogenate. Once the DNA is released from
the nucleus, it must be protected from nucleases, enzymes which will degrade the DNA.
Keeping the cell homogenate cold and various chemical components of the homogenization
medium help restrict the action of these nucleases. The final step of this protocol involves
precipitation of DNA from the homogenate. When the homogenization medium is added in
the first step, the positive ions of the salts (sodium chloride and sodium citrate) bind to the
negatively charged DNA backbone, creating a DNA molecule with a neutral charge. When a
cold polar solvent, like ethanol, is added to the DNA solution, the DNA is precipitated out of
solution, leaving other cell components behind (proteins, lipids, polysaccharides, general cell
debris. (SIM, 2020)

Material and Methods

Materials: Onions, knives, chopping boards, gloves. Beakers, Blenders, Filters funnel and
filter paper. Ice baths, water bath at 600C. 15-20ml of ice cold ethanol per group, pasteur
pipette or glass rods, methylene blue. Cover slips / slides

The procedure consisted in several steps, but it was taken into consideration some deviations
that took place, such as temperature and time modifications.

 The onion was chopped.

 Lyisis buffer solution (100 ml) was put into a 250 ml beaker and heated at
approximate 520C in a water bath.
 50 g of chopped onion was added to lyisis buffer solution, then stirred and let sit for
15 min. at about 530C
 The beaker was removed from the water bath and immediately placed on ice for five
minutes.
 The mixture was then put into a blender.
a) Blended at low speed for 45 seconds and stop.
b) Blended at high speed for 30 seconds and stop.
 The mixture was poured onto a piece of cheesecloth folded in four and draped over a
500 ml beaker that was already on ice.
 The filtrate was gently squeezed into a graduated cylinder, and was put on ice for
another 15 minutes.
 15-20ml of ice cold ethanol was added into the graduated cylinder by pouring it
slowly. A layer of ethanol was formed on top of the onion filtrate.
  The tube was let to sit for 3 – 5 minutes without disturbing it. Bubbles were formed
and DNA was precipitated out the solution. The DNA became visible as white strings
in the ethanol layer.
 DNA was spooled by snagging it with a Pasteur pipette or glass rod.
 A small amount of DNA was put on slide. A drop of methylene blue was added. A
cover slip was put. Observation and drawings have been made.

Results

Results of the experiment showed a clear layer of ethanol, that was on top of the onion
filtrate. The ethanol was used to precipitate the DNA. In water, DNA is soluble. When it is in
ethanol, it uncoils and precipitates leaving behind the other cell components that are not
soluble in ethanol. Therefore DNA precipitated out of the solution becoming visible as white
strings in the ethanol layer.

Discussions

For isolation of onion’s DNA, heating and blending was performed, in order to
disrupt the cells. The lyisis buffer solution was firstly heated in order to weaken the
phospholipid layer in the cell membrane and to denature the DNases enzymes. The sample
was also blended in order to disrupt the cell wall, nuclear membrane and cell membrane.
The results of the experiment, confirmed that DNA was isolated, this appearing in the
form of a stringy substance bubbling out of the solution. The presence of DNA was
confirmed by gently picking DNA filaments on a glass slide, adding methylene blue and
using a cover slip. It is known that the homogenizing solution contains a detergent which
emulsifies proteins and lipids, binding the cell membrane together. Therefore, the detergent
made them to precipitate out the solution. Ethanol was added to the beaker to make the
composition in the filtrate to stay in the solution. The DNA was present in the interface
of the solution.
In order to extract pure DNA, UV spectroscopy is used. This technique gives the
absorbance of the sample. The absorbance ration at 260nm and 280 nm was used to
measure the purity of DNA. If the ratio at 1.8 is obtained then the DNA is pure, and if
the ratio is lower than 1.8, then DNA may not be pure. This is because of the presence of
proteins, other contaminants and wrong measurements.
The recipe for onion DNA isolation calls for the use of lyisis buffer solution to break down
some of the remaining proteins in the solution before extracting DNA, and points out that
pineapple juice, which contains bomelain, will make an acceptable alternative (as it also
contains enzymes that break down proteins).

Conclusion
In the result of this experiment, DNA of the onion was isolated. This appeared in the form of
precipitate. DNA filaments have been extracted out the precipitate solution and further DNA
of the onion was observed under the microscope.
Bibliography
Encyclopaedia Britannica. (2020). Retrieved February 10, 2020, from
https://www.britannica.com/science/DNA

Genetics home reference. (2020). Retrieved February 13, 2020, from

https://ghr.nlm.nih.gov/primer/basics/dna

SIM, W. C. (2020). https://docplayer.net/21322835-Dna-spooling-1-isolation-of-dna-from-

onion.html. Retrieved February 10, 2020, from https://docplayer.net:
https://docplayer.net/21322835-Dna-spooling-1-isolation-of-dna-from-onion.html

Sirakov, I. N. (2016, March 16). Nucleic Acid Isolation and Downstream Applications. Retrieved
February 12, 2020, from https://www.intechopen.com:
https://www.intechopen.com/books/nucleic-acids-from-basic-aspects-to-laboratory-tools

What is biotechnology. (2020). Retrieved February 10, 2020, from

https://www.whatisbiotechnology.org/index.php/science/summary/extraction/dna-extraction-
isolates-dna-from-biological-material