MARWA ABOUARRA
YILDIZ TECHNICAL UNIVERSITY
DEPARTMENT OF B IOMEDICAL
E NGINEERING
EXPERIMENT
6
DNA ISOLATION & GEL ELECTROPHORESIS
Author:
MARWA
ABOUARRA
14.December.2022
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C o n t e n t s:
1. Purpose of the Experiment
………………………………………………………page 3
2. Theory
………………………………………………………page 3-4
3. Materials
………………………………………………………page 5
4. Method
………………………………………………………page 5-6
5. Results
………………………………………………………page 6
6. Discussion
…………………………………………………….... page 7-8
LIST OF FIGURES
FIGURE 1. BASIC METHOD FOR DNA EXTRACTION (BARTEE L., 2017)...........................................................................3
FIGURE 2. GEL ELECTROPHORESIS SYSTEM ...................................................................................................................4
FIGURE 3. PICTOGRAPH OF ALL NECESSARY COMPONENTS FOR GEL ELECTROPHORESIS. ................................................4
FIGURE 4.THE SAMPLES AFTER ADDING THE BLUE DYE AND PIPETTING THEM ON THE PAPER WITH 2-20 UL PIPETTE ......6
FIGURE 5. CASTING STAND WITH TRAYS AND COMB(THE WHITE PIECE) ........................................................................6
FIGURE 6.ELECTROPHORESIS SYSTEM ............................................................................................................................6
FIGURE 7. CLOSER PIC OF THE RESULTS UNDER THE UV LIGHT ........................................................................................6
LIST OF TABLES
TABLE 1. MATERIALS USED IN THE LAB ..........................................................................................................................5
TABLE 2. RESULTS ..........................................................................................................................................................6
REFERENCES
[1] Medline Plus. What is DNA? medlineplus.gov. Published January 19, 2021.
[2] Bartee L. DNA Isolation, Gel Electrophoresis, and PCR. Pressbooks.pub. Published 2017.
[3] The Wolbachia project. Lab 4: Gel Electrophoresis MiniOne Project Guide.; 2021.
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1.
Purpose of the Experiment
DNA fragments of various sizes will be separated using agarose gel electrophoresis.
2.
Theory
Humans and nearly all other species carry their genetic information in DNA, also
known as deoxyribonucleic acid. The DNA of an individual can be found in almost
all of their cells. The cell nucleus contains the majority of the DNA. Adenine (A),
guanine (G), cytosine (C), and thymine (T) are the four chemical bases that make up
the code that stores the information in DNA. The information available for
constructing and maintaining an organism depends on the order, or sequence, of
these bases. DNA nucleotides link up to form units referred to as base pairs, A with
T and C with G. A sugar and phosphate molecule are also joined to each base. A
nucleotide is a base, sugar, and phosphate compound that comes together to form a
double helix shape (Medline Plus, 2021).
In order to study or work with nucleic acids, cells' DNA must first be extracted.
Different methods are employed to extract various types of DNA (Figure 1). The
majority of nucleic acid extraction methods involve breaking down the cell first,
followed by the application of enzymatic processes to eliminate all undesirable
macromolecules, such as proteins and other possible contaminants. Using a detergent
solution that contains buffering agents, cells are cracked open. Enzymes are used to
inactivate macromolecules like proteins and RNA to stop breakdown and
contamination. Alcohol is subsequently used to extract the DNA from the solution.
Because the resultant DNA is composed of lengthy polymers, it takes the form of a
gelatinous mass. All of the nucleic acid in a cell is extracted using this technique.
This comprises both RNA and genomic DNA (all of the DNA found in the genome)
(Bartee L., 2017).
Figure 1. Basic method for DNA extraction (Bartee L., 2017)
The quantity and molecular weight of the DNA that must be used, the purity
needed for subsequent applications, as well as the cost and time involved, all
influence the method that is chosen.
DNA fragments can also be separated using the gel electrophoresis technique by
passing them across an agarose gel, which resembles Jello. Agarose gels, which are
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derived from a seaweed polysaccharide, generate tiny pores that serve as sieves to
segregate DNA according to size; smaller DNA molecules pass through the pores
more quickly and easily than bigger molecules. To enable accurate insertion of PCR
products into the gel, loading wells are positioned at the top of the gel. DNA
molecules that are negatively charged are moved by an electrical current from a
negative electrode (-) to a positive electrode (+). DNA moves across the gel in a
single, vertical line. The voltage of the electrical field, the amount of agarose
present, and—most significantly—the size of the DNA molecule all has an impact
on the speed of the movement. An agarose gel cannot reveal DNA by itself. As a
result, a fluorescent stain that binds to DNA and fluoresces under UV or blue light is
added to the gel. On the agarose gel, DNA will show up as a horizontal line or band
(The Wolbachia project, 2021).
Figure 2. Gel Electrophoresis System
Figure 3. Pictograph of all necessary components for gel electrophoresis.
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3.
Materials
Table 1. materials used in the lab
Devices
Chemicals
Consumables
100-1000µL pipette
DNA G1
Pipette tips
Power supply
Ethanol 7.9t
Whole blood
Gel electrophoresis
system
Lysis solution
Thermo scientific GeneJET genomic DNA
purification column & collection tube
Vortex
Wash buffer 1
Fume cupboard
UV camera
Wash buffer 2
1.5 mL microcentrifuge tubes
Gel Tank
Elution buffer
Minicentrifuge
2-20µL pipette
Agarose
Beaker
10XTris/Acetate/EDTA
buffer (TAE )
DNA loading dye
4.
Method
4.1 DNA Isolation
Using the 100-1000µL pipette and changing the tips at each step of
loading a different solution like always:
i.
400 μL of Lysis Solution added to 200 μL of whole blood,
ii.
The assistant then added 20 μL of Proteinase K Solution to the 200 μL of
whole blood and lysis solution,
iii.
Mixed using the pipetting technique to obtain a homogenous suspension.
iv.
The sample was incubated at 56 °C with intermittent vortexing until all of
the cells had been lysed (10 min),
v.
200 μL of the given ethanol was added and mixed by vortexing
vi.
Transferred the ready lysate to the collecting tube with a GeneJET
Genomic DNA Purification Column attached,
vii.
Centrifuged the column at 6000 x g for 1 minute.
viii. The ripple solution's container, the collection tube, was discarded.
ix.
The GeneJET Genomic DNA Purification Column was then placed inside
the empty DNA centrifuge,
x.
500 μL of Wash Buffer I was added,
xi.
The flow-through was removed after the purification column had been
centrifuged for 1 minute at 8000 x g and then put back into the collecting
tube,
xii.
Added 500 μL of Wash Buffer II to the GeneJET Genomic DNA
Purification Column,
xiii. Centrifuged for 3 min at maximum speed (≥12000 x g),
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xiv.
xv.
xvi.
xvii.
200 μL of Elution Buffer was added then to the center of the GeneJET
Genomic DNA Purification Column To elute genomic DNA,
Centrifuged for 1 minute at 8000 x g after 2 minutes of room temperature
incubation,
The filtration column was thrown away.
Utilize the purified DNA right away in subsequent applications.
4.2 Agarose Gel Electrophoresis
i.
1X TAE and agarose gel was prepared by the assistants because
Ethidium bromide is extremely poisonous/toxic and a potent mutagen.
The right safety measures are required by a pro.
ii.
Loading of the DNA samples
To prepare sample for electrophoresis:
a. 2 μl of 6X gel loading buffer added to 10 μl of DNA sample by the
assistant
b. Mixed well by pipetting then,
c. The sample was loaded onto the well, the rest of the students did
them, same as what the assistant did.
d. The control DNA then was loaded, after extracting the DNA sample
iii.
Electrophoresis
i.
According to conventions, they attached the power cord to the
electrophoretic power source: Black(-) cathode and red(+) anode.
ii.
Depending on the size of the DNA to be seen, electrophorese at
100–120 volts and 90 mA until dye markers have migrated a
suitable distance.
*anything that had whole blood was thrown away in the trash including pipette tips,
gloves and centrifuge that had the whole blood.
5.
Results
Table 2. results
Figure 4.the samples after adding the
blue dye and pipetting them on the
paper with 2-20 ul pipette
Figure 5. Casting Stand with
Trays and Comb(the white
piece)
Figure 6.Electrophoresis system
Figure 7. closer pic of the results
under the uv light
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6.
Discussion
Thermo Scientific's GeneJET genomic DNA purification column tube is made to quickly
and effectively separate high-quality genomic DNA from a variety of mammals cell
culture and tissue samples, whole blood, bacteria, and yeast. The design makes use of a
practical spin column that is based on silica-based membrane technology. Specimens are
digested with Proteinase K in either the provided Digestion or Lysis Solution depending
on the starting material. Following the addition of ethanol, the lysate is poured onto the
purification column, where the DNA adheres to the silica membrane. By using the readymade wash buffers to wash the column, impurities are successfully eliminated.
The Elution Buffer is then used to elute genomic DNA under low-ion strength conditions.
Gel electrophoresis is a technique for length-based separation of molecules, including
DNA strands and proteins.
The electrophoresis chamber's electric current is created by the power source.
The TAE buffer solution is used to help carry an electric current.
On the agarose gel, shorter DNA strands move farther and more quickly than longer ones.
Observe that when an electric current is supplied, the positive electrode is furthest from
the wells and the negative electrode is closest to them.
The largest fragment will be found closest to the well where it began because it will move
slower than the smaller fragments, which can move through the gel easier.
Two main components of loading dye are
i.
a visible dye that shows how far the DNA has traveled on the gel and
ii.
glycerol, which is denser than buffer and keeps samples from floating
back out since it is heavier than the buffer.
A DNA ladder is a collection of pieces of DNA that have specific diameters. The ladder,
also known as a DNA marker, is loaded next to test samples as a guide for determining
band size.
DNA can move through the agarose gel thanks to the conductivity of running buffer.
Making the agarose gel with the same buffer is crucial.
While preparing for the DNA isolation If there are any signs of leftover solution in the
purification column, the collecting tube should be drained, and the column should be
spined once more for one minute at its highest speed. Then the GeneJET Genomic DNA
Purification Column can be transferred to a sterile 1.5 mL microcentrifuge tube and the
collection tube containing the flow-through solution can be thrown away.
DNA Ladder : Since all the bands are visible on the gel and the ladder that means the
samples were well loaded in this experiment.
DNA Stain is a colorant that attaches to DNA and makes it visible under UV or blue light.
Lane: The vertical DNA movement route that runs beneath each loading well.
Loading Dye: A loading dye is added to an agarose gel to help with loading. Glycerol and
colored dye are both present.
An agarose gel indentation where samples are loaded is called a loading well.
Negative control: is intended to have an adverse effect and ensure that the process and
samples are not compromised.
Positive Control: A known variable that should produce the desired outcome.
A conductive liquid called a running buffer enables DNA to move through an agarose gel.
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