MOTLHOIWA LUCKY LESLEY

223037369

15 APRIL 2024

BCH 212 PRACTICAL 7

TITLE

Isolation of plant Deoxyribonucleic acid from onions

INTRODUCTION

Electrophoresis is a technique used to separate DNA fragments (or other

macromolecules, such as RNA and proteins) based on their size and charge. Using an
electric field, molecules (such as DNA) can be made to move through a gel made of agar
or rage at one end which pushes the polyacrylamide (Lee and Bahaman, 2012). The
electric field consists of a negative charged molecule through the gel, and a positive
charge at the other end that pulls the molecules through the gel. The molecules being
sorted are dispensed into a well in the gel material. The gel is placed in an
electrophoresis chamber, which is then connected to a power source. When the electric
current is applied, the larger molecules move more slowly through the gel while the
smaller molecules move faster. The different sized molecules form distinct bands on the
gel (Gonsebatt, 1996).

The term” gel” in this instance refers to the matrix used to contain, and then separate the
target molecules. In most cases, the gel is a cross-linked polymer whose composition and
porosity are chosen based on the specific weight and composition of the target to be
analyzed. According to (Muhammed et al., 2020) when separating proteins or small
nucleic acids (DNA, RNA, or oligonucleotides) the gel is usually composed of different
concentrations of acrylamide and a cross-linker, producing different sized mesh networks
of polyacrylamide. When separating larger nucleic acids (greater than a few hundred
bases), the preferred matrix is purified agarose. In both cases they form a solid, yet
porous matrix. Acrylamide in contrast to polyacrylamide is a neurotoxin and must be
handled using appropriate safety precautions to avoid poisoning.

Agarose is composed of long unbranched chains of uncharged carbohydrates without

cross links resulting in a gel with large pores allowing the separation of large molecules
and smaller molecule complexes (Lrvine and Yashida, 2000).Electrophoresis refers to
the electromotive force (EMF) that is used to move the molecules through the gel
matrix, by placing the molecules in wells in the gel and applying an electric field, the
molecules will move through the matrix at different rates, determined largely by their
mass when the charges are the same then, the electrical field generated by the
electrophoresis procedure will affect the species that have different charges and therefore
will attract the species according to their charges being opposite. Species that are
positively charged (cations) will migrate towards the cathode which is negatively charged.
If the species are negatively charged (anions)they will migrate towards the positively
charged anode (Mandriol and Blackman, 2006).

AIM

The of this experiment was to isolate DNA from an onion and to discover the fundamental
properties of DNA and the scientific methods to isolate it.

MATERIALS AND METHODS

Isolation of DNA

A 100 ml graduated cylinder was used to fetch 80 ml of distilled water, 10 ml graduated

cylinder was used to pour 10 ml of dishwashing liquid (Sunlight liquid) and then a weighing
stand was used to measure the 10 g of non-iodized salt. Before weighing non-iodized
salt, the weighing boat was inserted into the weighing stand and its mass was put to 0.
Then 80 ml of distilled water, 10 g of dishwashing liquid and 10 g of non-iodized salt were
poured into the 250 ml beaker. The amount of weighed 50 g of finely grated onion was
added to the solution. Then the solution was incubated in a 60 Degree Celsius water bath
for 15 minutes. The homogenate was filtered through a four-layer cheese cloth and the
filtered solution containing DNA was saved. The amount of weighed 1 g of meat tenderizer
was added to the strained homogenate and then it was left to stand for 5 minutes at room
temperature. After the homogenate was cooled at 10°C on ice. Very slowly 50 ml of ice-
cold isopropanol was added by pouring it down the side of the titled homogenate on the
bottom. It is essential that the isopropanol and homogenate form separate layers with the
homogenate on the bottom. The stringy white DNA that appears at the interface was
spooled out by swirling a glass rod around at the isopropanol/homogenate interface. The
rod was always turned-on in the same direction. The DNA looked like a blob of mucus on
the glass rod. The DNA was then suspended in 500 µl TE.

Preparation of a 0.8% agarose gel

A 500 ml graduated cylinder was used to measure 495 ml of distilled water, then the water
was poured into a bottle and 5 ml of TAE was added in the bottle containing distilled water
to create a 0.5 M of TAE buffer. Then 50 ml TAE buffer was poured into an Erlenmeyer
flask and 0.4 g of powdered agarose was added to the Erlenmeyer flask which contained
the TAE buffer. Then the mixture was heated using a microwave for about 20-60 seconds.
Heating the slurry for the minimum time was to allow all grains of agarose to dissolve.
The solution was left to cool to an approximate temperature, 60°C. Ethidium bromide was
added to a final concentration of 0.5 µl/ml (2.5 µl of a stock solution of 10 mg/ml in water)
and mixed well.

Evaluation of DNA integrity by agarose gel electrophoresis

After the gel was completely set, the comb and the rubbers that were covering the edges
of the plastic tray were carefully removed and mounted the gel in the electrophoresis.
Enough electrophoresis buffer was added to cover to a depth of about 1 mm. In a sterile
microcentrifuge tube, a sample of isolated DNA was mixed with gel loading buffer. A
micropipette was used to slowly load 30 µl of the mixture into the slots of the submerged
gel. A sample of ƛ DNA was loaded with Pstl restriction endonuclease (10 µl containing
approximately 1 µg DNA). That provided the ladder of DNA bands of defined sizes as
molecular markers for determining the size of your DNA. Then the lid of the gel tank was
closed, and the leads were attached so that DNA migrates towards the anode (red label).
Voltage of 1-5 V/cm was applied (measured between as the distance between the
electrodes; normally 50-100 V). Then it was electrophoresed until the bromophenol blue
had migrated three-quarters the length of the gel, approximately for 45 minutes. Then the
electric current was turned off and the leads were removed and lid from the gel tank. Then
the gel was examined using an ultraviolet (UV) illuminator.
RESULTS

Figure 1: A representative gel electrophoresis profile of UV illuminator. Genomic DNA

isolates from selected onion. Lane 1: molecular weight marker (100bp); lane 2: positive
control; lane 3: positive control; lane 4: positive isolates Genomic DNA.

ANSWERS

a) It is because it reduces surface tension and prevents bubbles and uneven gel formation

b) Sodium laurel sulfate

c)Because it prevents DNA degradation and promotes DNA precipitation.

d)It’s to break down the protein present in the sample and the reagent is Proteinase K.
DISCUSSION

The 10 ml of dishwashing liquid was added to the initial solution to lyse the tough cell wall
of the cell or break down the cell membrane and release the cytosol. Sodium laurel
sulphate can be used as a place of dishwashing. The salt solution helps the DNA separate
(Zhang and McGown, 2016). The salt shields the negative ends of the phosphates of
the cell membrane so by adding salt, we help neutralize the DNA charge and make the
molecule less hydrophilic, meaning it becomes less soluble in water. Salt also helps to
remove proteins that are bound to the DNA and to keep the proteins dissolved in the
water. Meat tenderizer powder contains enzymes known as proteases (or proteolytic
enzymes) that break down peptide bonds between amino acids found in proteins. DNA is
surrounded by different types of proteins so by breaking the bonds that hold those
proteins together, DNA will become more accessible. Bromelain is normally used in the
place of meat tenderizer during DNA isolation protocols.

In this experiment the results of DNA integrity by agarose gel electrophoresis were
observed. When several samples are placed into neighboring(adjacent) wells in the gel,
they will run in parallel in separate lanes. Each lane demonstrates separation of the
components from the original mixture as one or more separate bands, one band per
component, depending on the number of individual molecules. Incomplete component
separation might result in overlapping bands or indistinguishable blurs reflecting many
unresolved components. Bands in various lanes that finish up at the same distance from
the top include molecules that traveled through the gel at the same rate, implying that
they are roughly the same size. There are molecular weight size markers available that
comprise a combination of recognized sized molecules. If such marker was run on one
lane in the gel parallel to the unknown samples, the bands observed can be compared to
those of the unknown to determine their size.

The buffer was in charge of transporting charges through the agarose gel. Additionally, it
was discovered that DNA molecules moved at different speeds and distances in the
agarose gel depending on the charges and sizes that each molecule carried. The length
of a band is roughly inversely related to the logarithm of the molecule's size. Techniques
for electrophoresis have several limitations (Chery et al., 2006). Gels may melt during
electrophoresis because running electricity through them heats them up. The distance a
band travels is approximately inversely proportional to the logarithm of the size of the
molecule. There are limits to electrophoretic techniques. Since passing current through
a gel causes heating gels may melt during electrophoresis. Electrophoresis is
performed in buffer solutions to reduce pH changes due to the electric field, which is
important because the charge of DNA and RNA depends on the pH, but running for too
long can exhaust the buffering capacity of the solution. Further different preparations of
genetic material may not migrate consistently with each other, for morphological or other
reasons. The DNA molecule moved in the direction of anode from the cathode since the
agarose gel was negatively charged. The buffer was the one that was responsible to carry
charges in the agarose gel. It was also found that DNA molecules has migrated different
distances in the agarose gel because of the different charges that they carried and the
size that each molecule has.

The small molecule travelled a long distance as compared to those which has big size.
So, this means that the bigger the size of the molecule the shorter the distance it will
travel and the smaller the size of the molecule the longer the distance it will travel. The
molecule when it is big the electrophoretic mobility is low and when the molecule is small
the electrophoretic mobility is high. Genomic DNA contains all the genetic, chromosomal
material of an organism and it encodes all the components necessary for life. The purpose
of genomic DNA extraction is to separate this genetic material from the rest of the cell.
REFERENCES

Ammam, M., 2012. Electrophoretic deposition under modulated electric fields: a

review. Rsc Advances, 2(20), pp.7633-7646.

Chéry, C.C., Moens, L., Cornelis, R. and Vanhaecke, F., 2006. Capabilities and limitations
of gel electrophoresis for elemental speciation: A laboratory's experience. Pure and
Applied Chemistry, 78(1), pp.91-103.

Dr D. Stewart Lrvine and Jererny P Yashida (2000), journal of andrology, volume 21, issue
1, page 33.

Gain Carlo manicard, mauno mandriol and roger L. blackman (2006) biological review,
volume 6, page 890-898.

Guillermo Elizondo, and maria E. Gonsebatt (1996), gel electrophoresis, volume 307
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Lee, S.V. and Bahaman, A.R., 2012. Discriminatory power of agarose gel electrophoresis
in DNA fragments analysis. Gel electrophoresis-principles and basics, pp.41-56.

Muhammed, A.Q., Maraie, N.K. and Al-Sudani, B.T., 2020. Efficacy of gel electrophoresis
for proteins and biotechnological products–an overview. Al Mustansiriyah Journal of
Pharmaceutical Sciences, 20(4), pp.45-56.

Zhang, X. and McGown, L.B., 2016. Sequence‐based separation of single‐stranded DNA

at high salt concentrations in capillary zone electrophoresis. Electrophoresis, 37(14),
pp.2017-2024.