Name: İlayda KIRAN
Date: 30.11.2021
Student’s ID: 280104037
Assistant’s Name: Azime AKÇAÖZ
DNA Isolation and Agarose Gel Electrophoresis
Abstract
In this lab, a comprehensible technique for DNA isolation and agarose gel electrophoresis
were done to observe DNA itself and its fragments. The main idea that underlies this study is
to examine how to isolate DNA from various individuals and species, to make DNA ready for
observation, to determine an agarose solution of convenient concentration according their needs
and to learn how to use electrophoresis for separating those DNA samples. At the end of this
process of isolation and seperation, different portions of DNA were observed by separating
from each other under a simple process of electrophoresis. The image occurred under the UV
light represents DNA portions that are dyed to be seen and their distribution into the agarose
gel according to their size.
Introduction
Process of DNA isolation is very important that most of the genetic studies rely on one’s
ability to isolate DNA from many, thus, scientists developed several isolation methods over
years. In this lab, three methods for DNA isolation are examined: Salting-Out, Organic
Extraction and Cesium Chloride Density Gradient. Salting-out is a simple technique for
separating the proteins and other wastes from the cell using high concentration of salts. Organic
extraction is a method where the cell is broken down by a detergent and mixed with organic
molecules such as phenol and chloroform to remove the wastes from the cell. Cesium chloride
density gradient is a complex process used for the separation of DNA where the cells lysed by
a detergent and mixed with CsCl and EtBr. However, EtBr is removed by isopropanol after
centrifuge and DNA is recovered by ethanol. All these methods serve a special purpose and are
used in different conditions. In this case, the most advantageous method can be considered as
salting-out method as it provides DNA with high purity and yield that can be used in
downstream applications, even though it may be unproductive method. Furthermore, organic
extraction provides DNA in aqueous phase but is a time-consuming process and contains such
high amount of alcohol waste during the extraction which sometimes causes the enzymes that
act during the PCR to be inhibited. In addition, this method makes DNA inadaptable to most of
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the later applications. Cesium Chloride Density Gradient provides high-quality DNA but is also
a time-consuming and expensive process which makes it useless for routine experiments.
Moreover, this method uses toxic chemicals which may cause harms unless the experimenter
taken good care. (1)
Agarose gel electrophoresis (AGE) is a technique used for analyzing nucleic acids. In this
technique, DNA fragments are separated according to their size. Agarose gel electrophoresis is
a widely used technique in most of genetic research due to its ability to supplying DNA.
Agarose gel electrophoresis consist of two main part which are agarose and electrophoresis (1).
Agarose is a polysaccharide refined from seaweed. It consists of two fundamental units that are
D-Galactose and 3,6-anhydrol-galactopryanose. Agarose is a powdered form of sugar before it
is suspended with a buffer solution. When agarose mixed with a buffer is first boiled. Next, it
poured into the casting tray and left to cool. During this cooling process, agarose undergoes a
cross-linking (H-bonding) which technically affects the pore size of agarose gel and is a weak
interaction bonds allowing agarose gel to stay in a flexible, gelatin-like form. Furthermore,
agarose gel electrophoresis is a process in which DNA is separated according to its charge and
size. This mechanism consists of electrical field and gel where DNA is located. Negatively
charged DNA begins to move towards positive electrode (red). In this case, there are some
factors affecting migration of DNA that can be listed as: 1) Agarose concentration, 2) The
strength of electrical field or voltage applied, 3) Size of DNA and 4) DNA conformation. There
are some commonly used gel running buffers such as EtBr, TAE which will be explained in
this lab. Tris-acetate-ethylenediamine tetra-acetic acid (TAE) is one of the most common
buffers used in agarose gel that consist of 40mM Tris, 20mM acetic acid and 1mM EDTA
(ethylenediamine tetra-acetic). This buffer, mainly, step in the enzymatic reaction and protects
DNA from this enzymatic activity. Ethidium Bromide (EtBr) is a harmful chemical that binds
to DNA and allows it to be seen under the UV light. Furthermore, DNA ladders are weight
markers used during this process which estimates the size of fragments. Finally, loading dye
that makes DNA visible under the UV light. (2)
Method
This lab can be fundamentally separated into two parts. In first part the isolation of DNA is
explained and in the second part the Agarose gel electrophoresis is explained. In the first part,
we used mortar-pestle technique and homogenized DNA from a plant. First, we smashed the
plant in a cup with adding it 10µl CTAB which is a detergent. Next, using a micropipette,
1000µl of liquid taken into an eppendorf safe-lock tube. Then we heated these tubes at 60-65oC
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for an hour. Tubes are transferred to centrifuge and for 10 minutes they centrifuged at
14,000rpm. Next, the supernatant taken to another Eppendorf tube and mixed with 500µl of
cold isopropanol. These tubes are taken to the centrifuge again at 14,000rpm for 5-6 minutes.
Then tubes are taken from the centrifuge and supernatant removed from the tube. 500µl of 70%
Ethanol solvent added on to the precipitate that involves DNA. Tubes are taken to the centrifuge
at 14,000rpm for 3-4minutes. When tubes are taken from the centrifuge the ethanol is taken
from the tubes carefully and left lid-open for ethanol to evaporate for a little time in case there
is still ethanol left in the eppendorf. Finally, 30µl water added on to the precipitate and tubes
were stored at -20oC for a week.
In the second part, we prepared 50ml agarose gel 1,5% of agarose using 750mg (0.75g) of
agarose. It can be calculated as the following steps:
1. 50x (1.5/100) = 0.75ml (agarose amount in total gel)
2. 0.75ml = 0.75g
Next, we added 50ml TAE buffer to the agarose and boiled until it gets clear. Never over-boil
the gel because it may cause harms both on the casting tray and to the experimenter. Next, when
the gel cooled to about 50oC, we added 2,5µl EtBr which can be simply calculated as:
1. If 1ml contains 0.5µg/ml EtBr, 50ml must contain 25µg/ml EtBr
2. 25µg is equal to 0.025 mg EtBr
3. In order to make our density 10mg/ml, 10=0.025/x where x equal to 0.0025ml.
4. 0.0025ml is equal to 2,5µl, hence, 2,5µl EtBr.
Next, we poured the gel into the casting tray and located the combs in the gel and left it to cool
until it takes the solid-like texture on the surface. Finally, we removed the combs and placed
the DNA samples in the gel.
Result
A: Genomic DNA
B: 28S rRNA
C: 18S rRNA
Smears appear on the gel.
D: tRNA
Figure 1. Genomic DNA isolation result of plant, Agarose Gel
Electrophoresis.
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The results of Agarose gel electrophoresis have shown in the figure 1. There are several
different molecules appear on the gel such as 28S rRNA, 18 rRNA, tRNA other than genomic
DNA. Various smears appear on the gel as well. Those are labeled and shown in the figure.
Discussion
DNA isolation is a commonly used process in genetic labs. We learned how to isolate DNA
from a plant by using several techniques. During this process, cell must be lysed to the nucleus
and even the nucleus must be broken down in order to obtain DNA. Several chemicals were
used to provide pure DNA, each which has specific responsibility in isolation. Starting with a
detergent which is technically used to lyse the cell membrane, we used CTAB. Because of its
chemical structure and the presence of so many polysaccharides on the cell wall, it is much
harder to extract DNA from plant cells than animal cells. The cell wall consists of hydrophilic
end groups and CTAB can dissolve these parts very well. In short, the main reason why CTAB
used in this experiment is that CTAB can handle such strong cell structure and destroy is much
easier comparing other ones. Thus, the feasibility of CTAB on isolation is higher than other
buffers. Intermediate steps such as centrifuge and heating-cooling are required conditions
and/or on account of making sure that the chemicals and enzymes in the mixture works well.
Next, we added isopropanol which allows DNA to precipitate from the solution. Isopropanol is
quite significant chemical necessary for DNA to be separated from the liquid phase for later
steps. Isopropanol removes hydration shell from the H2O molecules around the negatively
charged phosphate and DNA molecules come together as a result of this neutralization of
charge. Next, we added 70% ethanol solvent added to the solution. Ethanol removes
unnecessary or unwanted protein and carbohydrate in nucleic acid pellets. Ethanol is taken after
the centrifuge, as well as every other liquid was taken at the end of each step. Finally, water is
added to the precipitate. Water provides us a suspended version of DNA with no other
molecules.
We also examined and learned agarose gel electrophoresis. Depending on the results, there
are 4 bands unexpectedly occurred on the gel. Each band consist of different molecules with
different sizes. Band A involves the biggest molecules genomic DNA, whereas band D involves
the smallest ones tRNA. At the beginning of experiment, we expected to observe just one band
on the gel as we supposed to work only with the DNA. However, there were isolated RNA
types appeared on the gel, as well. This means our DNA solution that placed to the
electrophoresis chamber was not pure. If our DNA solution was pure, we would observe only
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the band A shown in the figure 1. An example for pure DNA vision under electrophoresis is
given in the figure 2. Our vision of DNA must be
E
D
A
B
C
something near to A, B or C, whereas our vision was
something looked like D and E. In other words,
experiment we made in this lab was not DNA specific,
thus, we must search for methods to prevent from
Figure 2. DNA samples visualized under UV light,
agarose gel electrophoresis. Pure DNA samples
formed only one band (A,B,C); samples involves
different kind of molecules forms more than one
band (D,E)
unnecessary molecules. To prevent from these bands,
hence, residual, or unwanted molecules such as RNA
types - or if pure DNA is required in any experiment,
we must add ribonuclease (RNase A) to the buffer we used during the isolation. TAE buffer,
obviously, does not damage these RNA types, hence, the existence of RNA is observed.
Furthermore, some other chemicals that harm RNA may be used during isolation process as
well. Back to out results, we know that the smallest molecules migrate the furthest. tRNA is
known as the smallest RNA type. DNA is the biggest molecule among these four, hence, the
size order is DNA>38S rRNA>18S rRNA>tRNA. However, these are not the only things that
appeared in the gel. Smears occurred during the visualization which is usually referred to as
improperly preparation of agarose gel. This can be explained simply as if gel is not poured
properly, it will not polymerize evenly which will cause molecules placed into the gel to smear.
Other reasons why smears appeared may be the overloading samples to the wells or damaging
the gel while loading the sample. If the gel moved after loading, it will cause the sample to spill
out of the well, hence, smear. To prevent from these smears, we must make sure that the process
is almost flawlessly continued and worked. While preparing agarose gel, some other buffers
can be used such as TBE. Tris-Boric-Acid EDTA is also very commonly known buffer used to
extract smaller/short fragments of DNA, whereas TAE provides bigger/long fragments of DNA.
Finally, according to all these data obtained, we must be able to say that this experiment did not
work as it is expected or needs to be modified at some points. However, overall experiment was
quite educational, obviously, for ones who used these two mechanisms for the first time.
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References
1. A. R. Hoezel (1998). Molecular Genetics and Analysis of Populations: A practical
Approach, Total DNA Isolation. (B. D. Hames, ed.), Footnote Graphics, Warminster,
Wilts, Printed in Great Britain.
2. Alfred Pühler, Kenneth N. Timmis. Advanced Molecular Genetics, 1.3
Characterization of Plasmid DNA by Agarose Gel Electrophoresis, 26-27. SpringerVerlag, Berlin.
3. Pei Yun Lee, John Costumbrado, Chih-Yuan Hsu, Yong Hoon Kim (April 20, 2012).
Agarose Gel Electrophoresis for Separation of DNA Fragments. Jove.com,
https://www.jove.com/t/3923/agarose-gel-electrophoresis-for-the-separation-of-dnafragments?text=agarose&
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