NAME : NOLUBABALO

SURNAME : NKOAYIDLI

STUDENT NUMBER : 202108442

COURSE CODE : BCH 211

TASK : PRACTICAL REPORT

EXPERIMENT : 13
Tittle: Isolation of plant DNA from unions.

INTRODUCTION:

DNA is deoxyribonucleic acid which is a polymer composed of two polynucleotide

chains that coil around each other to form a double helix carrying genetic instructions
for the development, functioning, growth and reproduction of all known organism and
many viruses. Ribonucleic acid (RNA) is a polymetric molecule essential in various
biological roles in coding, decoding, regulation, and expression of genes. DNA and
ribonucleic acid (RNA) are nucleic acids. Nucleic acids are composed of thousands
of basic units called nucleotides. RNA is made up of ribonucleotides and DNA is
made up of deoxyribose nucleotides. Alongside proteins, lipids, and complex
carbohydrates (polysaccharides), nucleic acids are one of the four major types of
macromolecules that are essential for all known forms of life. (Saengers,1984).

DNA extraction is a procedure of isolating the DNA from other cellular components
for the molecular or forensic analysis. Usually, a machine is used to extract DNA
from the cell that is called Bead Beater. It breaks the cell and extracts the DNA from
it. Gel Box is another machine which separates the sequences of DNA in the Gel. An
electric current is used for this purpose. In the end, centrifugation separates the DNA
from the solution through spinning. Extraction of DNA is an important step in the
diagnose use. The extracted DNA can be used for genetic testing. Genetic testing is
a technique used for the identification of the criminal. If there is a case of child
paternity, then also it helps a lot. (Elkins, 2013).

Electrophoresis refers to the electromotive force (EMF) that 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. Gel
electrophoresis is a method that separates macromolecules based on the size,
electrical charge, and other physical properties such nucleic acids or proteins. The
direction of movement is affected by the charge of the molecules, and the rate of
movement is affected by size, shape, the density of the gel and the strength of the
electric field. DNA is a negatively charged molecule, so it will move towards positive
pole of the gel when current is applied. (Hayes,1999).
TBE or EDTA is a buffer solution containing a mixture of tris base, boric acid, and
EDTA. In molecular biology, TBE and TAE buffers are often used in procedures
involving nucleic acids, the most common being electrophoresis. It used in agarose
electrophoresis typically for the separation of nucleic acids as DNA and RNA. TAE
(tris-acetate- EDTA) buffer is used as both a running buffer and in agarose gels. TAE
is used in denaturing gradient gel electrophoresis methods for broad-range mutation
analysis. TAE has been used at various concentrations to study the mobility of DNA
in solution with and without sodium chloride (Nacl), however, high concentration of
sodium chloride in a DNA sample retards its mobility. This may lead to incorrect
interpretation of the resulting DNA banding pattern. (Adams, 1987).

Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry,

molecular biology, genetics, and clinical chemistry to separate a mixed population of
macromolecules such as DNA or proteins in a matrix of agarose . Agarose is a sugar
matrix which the molecules are bind together and form a porous network like which
is the agarose gel. The pore size of the gel depends on the concentration of the
agarose gel. The results can be analysed quantitatively by visualizing the gel with
UV light and a gel imaging device UV illuminator depending on the type of analysis
being performed. (Ramsden,2000).

There are several applications of agarose gel electrophoresis: agarose gel

electrophoresis is used for molecular cloning; a construction of recombinant
molecules of DNA which are integrated into different organisms for genetic
modification purpose, it is used in studying people according to their distinct DNA
sequences, it helps to identify unknown samples and it is useful in the screening of
genetic disorders and the identification of protein abnormalities. Agarose gel
electrophoresis is also useful for genetic fingerprint. It identifies individuals based on
genetic code. It is used for forensic investigations, parentage testing and
genealogy.It is also used to separate the molecules of DNA and is useful during the
DNA manipulation procedure.(Brown,1993).
AIM: To isolate DNA from onions and evaluate the size of the DNA fragments using
the charge and density of DNA by the agarose gel electrophoresis and visualizing
the fragments using UV light.

METHODS AND MATERIALS:

1.ISOLATION OF DNA.

Eighty millilitre of water, ten millilitre of dishwashing liquid and ten gram of sodium
chloride (Nacl) were placed into a 250ml beaker. Fifty grams of finely grated onion
was added. The solution was incubated in a 60 ℃ water bath for 15 minutes. The
homogenate was filtered through a four-layer cheese cloth and the filtered solution
which contains DNA was saved. One gram of meat tenderizer was added to the
strained homogenate and then it was left to stand for five minutes at room
temperature. The homogenate was cooled to 10 ℃ on ice. Very slowly 50ml of ice-
cold isopropanol was added by pouring it down the side of the tilted beaker. the
white stringy DNA that appears at the interface was spooled out gently swirling a
glass rod around at the isopropanol/ homogenate interface. The rod was always
turned on in the same direction. The DNA were looked like a blob of mucus on the
glass rod. The DNA was resuspended in 500 μl TE.

2.PREPARATION OF 0,4% AGAROSE GEL.

The edges of a clean, dry, plastic tray supplied with the electrophoresis apparatus
were sealed with tape so as so form a mold. The mold was set on a horizontal
section of the bench. The amount of 0,4 grams of powdered agarose was added to
50 ml of TBE buffer in an Erlenmeyer flask. The neck of the flask was loosely
plugged with cotton wool. The slurry was heated in a boiling in a microwave oven on
full power for 20- 60 seconds. The slurry was heated for the minimum time required
to allow all the grains of agarose to dissolve. Wearing an oven glove the flask was
carefully swirled from time to time to make sure that any grain sticking to the well
enters the solution. The solution was cooled to approximately 60 ℃ and it was
cooled enough to hold. Ethidium bromide was added to the final concentration of 0.5
μg/ ml ( 2.5 μlof a stock solution of 10mg/ml in water) and mixed thoroughly. The gel
comb was positioned near one end of the mold and then approximately 40ml of the
warm agarose solution was poured into the mold. The gel was between 3mm and
5mm thick. The air bubbles were checked and not found. The gel was left to set
approximately 45 minutes.

3. EVALUATION OF DNA INTEGRITY BY AGAROSE GEL ELECTROPHORESIS.

After the gel was completely set, the comb was carefully removed and the tape and
mounted the gel in the electrophoresis tank. Electrophoresis buffer was added
enough to cover the gel to a depth of about 1mm. in a sterile microcentrifuge
tube(1,5ml), a sample of isolated DNA was mixed with gel loading buffer (10ml of 6 x
DNA loading buffer per 50ml DNA solution). Thirty microliter of the mixture was
slowly loaded into the slots of the submerged gel using a micropipette. A sample of γ
DNA restricted was loaded with Pstl restriction endonuclease (10 μl containing
approximately 1 μg DNA). That provided a ladder of DNA bands of defined sizes for
use as molecular size markers for determining the size of a DNA. The lid of the gel
tank was closed, and the leads were attached so that the DNA would migrate
towards the anode. The voltage of 1-5V/cm was applied (measured as the distance
between the electrodes; normally 50-100V). The bromophenol blue was
Electrophoresed until it has migrated three-quarters the length of the gel,
approximately 1 hour. The electric current was turned off and the lids were removed
and lid from the gel tank. The gel was examined using an ultraviolet (UV) illuminator.
The gel was photographed using the departmental gel documentation system
RESULTS:

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Genomic
DNA
100bp Wells
ladder

DNA
ladder

Figure 1: 0.8% agarose gel electrophoresis and the 100bp ladder after the gel was
exposed to ultraviolet light.

A. It dissolves the cell membrane, which is a liquid bilayer, and releases DNA
from the cell.

B. Shampoo was used as a commercial reagent in place of dishwashing liquid.

C. It is necessary to add salt to the solution because it helps to remove proteins
that are bound to the DNA and to keep the proteins dissolved in the water.
D. The function of meat tenderiser is to softens its fibres, improving its texture
and making it easier to chew and digest. Phenol is an organic solvent, so it is
not miscible with water and is used along with chloroform and isoamyl alcohol
for purification of the DNA to remove proteins and polysaccharide
contaminants.
E. The integrity of the purified DNA loads about 100 g per sample into a 0.8%
agarose gel and the size of the distribution is a suitable marker (e.g., the
important change we can make is our DNA slime). Increase the amount of
time the solution is immersed in ice so that it looks good or is easy to see.
DISCUSSION:

Initially the Sodium chloride salt was added because the positively charged sodium
ions from the salt shielded the negatively charged phosphate groups of the DNA
molecules, helping the DNA to precipitate in the solution. It also helps to remove the
protein that are bound to the DNA and to keep the protein dissolved in the solution
so that it does not precipitate in the alcohol along with the DNA. The detergent
dishwashing liquid degrades the membrane, phospholipids, and the protein to
release the DNA from the cells (Bruce,1993).

Grinding the onion breaks the cell wall of the membrane to permit release of the
DNA from the cell and allows the DNA to be seen clearly. Heating the mixture
degrades membrane phospholipids and proteins. Filtrations remove the residue of
the onion and collect the filtrate. Addition of meat tenderizer powder was because it
contains enzymes known as protease or proteolytic enzymes that breakdown
peptide bonds between amino acids found in proteins. DNA is surrounded by
different types of proteins so by breaking the bonds that hold these proteins together,
DNA will become more accessible by detecting a specific sequence on the DNA. As
the nucleic acids move large sequences of DNA will move slowly and stick to the gel
and cannot migrate further to the positive pores and smaller sequence will migrate
further.

Addition of isopropanol is less dense and moves up and causes the upper layer. The
DNA is the material in the clear alcohol and DNA is insoluble in isopropanol. Cooling
the mixture slows down the breakdown of the DNA which would occur if a high
temperature was maintained. In preparation of the agarose gel, it is heated in a
microwave because it is fast, and it does not evaporate and change the
concentration. The dye added to the agarose Ethidium bromide is for later to view
the nucleic acids fragments later. The comb was used to make wells to insert the
DNA samples. The coloured loading buffer added to the samples is composed of
glycerol and is used to increase the density and viscosity and causes the samples to
sink to the bottom and it also does not mix. DNA or RNA are negatively charged
because it contains a phosphate group, sugar ribose and nitrogen base and this
results the nucleic acids to move from the negative charge to the positively charged
electrode due to the attraction (Rogers,2010).

The dye in the DNA samples is used to keep track of the migrating samples. When
loading the samples, the pipette was parallel to reduce the possibility of accidentally
stubbing the walls of the wells. Passing electric current through the buffer and the gel
starts the process of separation of the nucleic acid fragments. Ethidium bromide
sticks to the nucleic acids and florax in the ultraviolet light using the illuminator. From
the results some of the sizes of the DNA samples are not visible this might happen
because the agarose gel was not fully dissolved. If not, the finish gel will have ridges
of different concentrations and the DNA nucleic acids will separate correctly. This
might have happened because the gel was set at an uneven surface and disturb the
concentration. The gel maybe damaged during inserting the DNA samples and it
causes the nucleic acid to migrate unevenly and some to dissolve in the buffer.
Addition of air bubbles along with the samples might have also disturb the results
and this causes samples to be lost in the wells and results in contamination. The
samples may have been loaded quickly and this might cause the sample to flow out
of the well. The comb might have been taken out before the set of the gel and affect
the shape of the wells.

The same experiment was conducted by Vysas S et.al 1998 at which they used
agarose gel electrophoresis to separate DNA fragments. During gelation agarose
polymers formed a network of bundle whose pore sizes to determine a gel’s
molecular sieving properties. The same common buffer TAE was used. After
separation, the resulting DNA fragments were visible as clearly defined bands. The
DNA fragments results were 765bp, 880bp and 1022bp along with a 2 log DNA
ladder. These results are different, and this may be caused by different concentration
of the agarose gel, they used 1,5% of agarose gel. Molecular serving is determined
by the size of pores generated by the bundles of agarose in the gel matrix. The
higher the concentration of agarose the smaller the pore size. Agarose gels are most
effective at the separation of DNA between 10bp and 25kb. Larger DNA fragments
are separated by the speed at which the reorient themselves with changes in current
direction (Craig,2010).
References:

Adams, D.(1987). Electrophoresis in agarose and acrylamide gels. Methods.pp.61-

87.

Bruce, A. (1993). Molecular Biology of the cell: Structure and Function of DNA.pp23.
Craig, C. (2010). Scientific Method Investigation: A step by step guide page 53.

Elkins, K.M. (2013). DNA Extraction. Forensic DNA Biology.pp.109(4):1045-50.

Rogers, S.O. (2010). Extraction of DNA from plant tissues. In plant molecular biology
manual. Springer, Dordrecht.pp.188-200.

Russel et al. (2001). Molecular Cloning: A Laboratory Manual.3 rd edition. Cold Spring
Harbor Laboratory Press. Cold Spring Harbor, NY.

Ramsden, E.N. (2000). Chemistry.4th edition. New York: Oxford University.

Saenger, W. (1984). Principles of Nucleic Acid Structure. New York: Springer-Verlag.