GENETIC ENGINEERING PRACTICAL REPORT

By :
Name : Mellya Rizki Pitriani
Student ID : B1B017031
Entourage : III
Group :4
Assistant : Heri Priyanto

MINISTRY OF RESEARCH, TECHNOLOGY AND HIGHER EDUCATION

JENDERAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGY
PURWOKERTO
2019
ISOLATION OF PLANT GENOME DNA

A. Aims

The aims of laboratory skill practical class is isolating the DNA of plant
genomes.
B. Materials
The material used in this practical class is DNA sample of leaf soybean (Glysine
max), CTAB (Cetyl Trimethyl Ammonium Bromide) Buffer, β-Mercaptoethanol,
CIAA (Chloroform Isoamyl Alcohol), ammonium acetate, isopropanol, etanol 70%,
TE (Triss EDTA), and alkohol 70%.
The tools used in this practical slas are micropippete, microtip, water bath, vortex,
centrifugator, microtube, PCR apparatus, electrophoresis apparatus, stationary and
camera.

C. Methods

The methods used in this practical class are:

1. Preparation Step
a. The CTAB extraction buffer and β-merchaptoethanol are heated in a water bath at
65oc for 1 hour.
b. Leaf samples were washed with water, sprayed with 70% alcohol and dried with a
tissue.
c. leaf samples are cut less than 1x1 cm.
2. The step of breakdown of cell walls
a. Samples are put into the kemortar and finely crushed, 1000μl CTAB buffer 10μl β-
merchaptoethanol added.
b. The results of the scour were incubated in a water temperature 65oC for 20
minutes, every 5 minutes in inversion.
3. The step of separating cell debris from DNA
a. The results of the scour were incubated in a water temperature 65oC and allowed
to stand for 2 minutes, then centrifuged 12000 rpm for 10 minutes.
b. The supernathan is taken and transferred to a new microtube, added cold CIAA
(24: 1) in a ratio of 1: 1 (supernathan: CIAA), homogenized by alternating, and
incubated on ice for 5 minutes.
4. The purification step of DNA from RNA and protein
a. The supernathan is taken and transferred to a new microtube, added cold CIAA
(24: 1) in a ratio of 1: 1 (supernathan: CIAA), homogenized by alternating, and
incubated on ice for 5 minutes.
b. The mixture was centrifuged at 12000 rpm for 20 minutes.
5. DNA precipitation step
a. Supernathan is taken and transferred to a new microtube, added cold ammonium
acetate as much as 1/10 the volume of supernathan, then added cold isopropanol as
much as 2/3 of the total volume (supernathan + ammonium acetate).
b. incubated in the freezer for 30 minutes then centrifuged at 13000 rpm for 30
minutes.
6. DNA washing step
a. The supernathan is removed, DNA deposits are added with 70% ethanol as much
as 500 ml and centrifuged at a speed of 13000 rpm for 5 minutes.
b. The supernathan is removed, the DNA deposit is dried by turning the tube over
tissue
7. Storage step
a. The DNA deposition was added with TE 1x as much as 50ml and allowed to
dissolve then stored in the freezer.
D. Result and Discussion

Figure 3.1 Visualization Result Of Isolation of Plant Genome DNA from

the Leaves of Soybean (Glysine max) in Entourage III.
 Details: (K+) Postive Control; (K1) Group 1 sample; (K2) Group 2
sample; (K3) Group 3 sample; (K4) Group 4 sample; (K5)
Group 5 sample. Source: Biology Molecular Laboratory
Documentation (2019).
Based on the results of the practicum and the changes made at this event, the
results of the visualization were in the form of parallel DNA bands. The visualization
sample showed that in group 3 and group 5 the samples showed the clear band and
comfortable based on positive control samples, while the results from group 2, 3 and
4 samples did not give clear results or there were smears on the electrophoresis
results. Parallels that use electrophoresis can compare with leaders or markers that
show base pairs of samples of DNA molecules. Electrophoretic visualization results
are obtained, namely smears because the visible bands are not too clearly bordered.
According to Rogers and Bendich (1994), several factors that can influence
electrophoresis results are DNA that has been degraded due to samples that take
longer or wrong times and DNA pellets that can be used in the storage of DNA from
DNA and protein samples of soybean leaves at the stage of cell wall splitting .
Another factor that also affects the results of smears or no bands on the
electrophoresis results is the pippetting process which when taking supernathan,
nathan located at the bottom of the microtube can be carried along with supernathan.
DNA isolation is a method used to separate DNA from cells, both from the
nucleus, mitochondria, and chloroplasts. The first stage in DNA isolation is the
process of destruction or destruction of membranes and cell walls. Cell splitting
(lysis) is the stage from the beginning of DNA isolation aimed at removing the
contents of cells. The destruction stage of a cell or tissue has several ways namely by
physical means such as grinding samples using mortar and pestle in liquid nitrogen
or by using the freezing-thawing and irradiation methods. Another way is to use
chemical and enzymatic. Chemical destruction such as the use of detergents that can
dissolve lipids in the cell membrane causing cell membrane destabilization. While
enzymatic methods such as using proteinase K to lyse membranes in blood cells and
degrade globular proteins or polypeptide chains in cell components (Surzycki, 2000).
The lysis process uses detergents, often using sodium dodecyl sulphate (SDS)
as the coating stage of the cell membrane. The detergent in addition to playing a role
in lysis of cell membranes can also play a role in reducing the activity of the
nuclelease enzyme which is a DNA degrading enzyme. SDS can also damage cell
membranes and cause chromosomal rupture. In addition to SDS use, other detergents
such as Cetyl Trimethylammonium Bromide (CTAB) are also often used to lyse cell
membranes in the isolation of plant DNA. CTAB is a method commonly used in
DNA extraction of plant genomes that contains polysaccharides and polyphenol
compounds. The use of a CTAB buffer as a substitute for liquid nitrogen for
extraction can produce quality DNA products. CTAB buffer can be used to isolate
DNA in plants. Good quality DNA extraction products are shown with DNA bands
that look thick and clean when visualized using gel electrophoresis images (Ardiana,
2009).
Impurities due to cell lysis are separated by centrifugation at moderate speeds
around 3000-5000 rpm for 5 to 10 minutes. The third stage is purification of DNA,
which aims to remove some contaminants such as secondary compounds (phenols),
polysaccharides, RNA and proteins. Purification of protein and RNA contaminants
was carried out using isoamilalkohol chloroform compounds, acetic acid, and RNAse
enzymes. Isoamilalkohol chloroform compounds and acetic acid function to
denaturate proteins chemically, proteinase K enzymes can be used to destroy proteins
and RNAse enzymes are used to destroy RNA so DNA can be completely isolated
(Muhammad and Praseno, 1991). Then the separated nucleotide molecules (DNA
and RNA) are cleaned from the remaining protein using phenol. In this process a
small portion of RNA can also be cleaned. While chloroform is used to clean up the
remnants of protein and polysaccharides from the solution. The process of
centrifugation with high speed will precipitate white flour (DNA) and stick to the
bottom of the ependorf tube (Tenriulo et al., 2001).
Precipitation (concentration) of DNA is done using cold isopropanol which
aims to make the DNA settle / collect at the same time separating it from the
remaining mineral salts of CTAB. The pellet produced by isopropanol is cleaned
using ethanol 70%. This purification is the most important step in DNA Isolation.
Because if there are contaminants other than DNA, the DNA isolation results are
considered to be failed. This contamination can reduce the quality of DNA from
isolation and result in invalid data obtained. In the use of CTAB buffer, other
reagents such as NaCl, EDTA, Tris-HCl and 2-mercaptoethanol are often added.
NaCl functions to remove polysaccharides while 2-mercaptoethanol functions to
eliminate the content of polyphenol compounds in plant cells. 2-mercaptoethanol can
remove polyphenols in plant cells by forming hydrogen bonds with polyphenol
compounds which will then separate from DNA. Polyphenol compounds need to be
removed in order to obtain good quality DNA. Polyphenols can also inhibit the
reaction of the Taq polymerase enzyme during amplification. The use of 2-
mercaptoethanol by heating can also denaturate proteins that contaminate DNA.
EDTA functions as a cell destroyer by binding to magnesium ions (these ions
function to maintain the activity of nuclelease enzymes that damage nucleic acids)
(Tenriulo et al., 2001).

REFFERENCES
Ardiana, W., 2009. Teknik Isolasi DNA Genom Tanaman Pepaya dan Jeruk dengan
Menggunakan Modifikasi Buffer CTAB. Buletin Teknik Pertanian, 14(1),
pp. 12-16.
Muhammad, S. A., and Praseno, 1991. Pengantar Kloning Gena. Yogyakarta:
Yayasan Essentia Medica.
Rogers, S.O. and Bendich, A.J., 1994. Extraction of total celluler DNA from plants,
algae, and fungi. Plant Mol Biol DI: pp. 1-81.

Surzycki, S. 2000. Basic Techniques in Molecular Biology. New York: Springer-

Verlag, Berlin, Heidelberg.
Tenriulo, A,, Suryati, E., Parenrengi, A., Rosmiat, 2001. Ekstraksi DNA Rumput
Laut Kappaphycus alvarezii dengan Metode Fenol Kloroform. Marina
Chimica Acta, 2(2), pp. 6-10.