Name of the author: Çağatay Utku ÇAMYAR

Date of the experiment: 21.12.2023

Submission date: 28.12.2023
Name of the experiment: Agarose Gel Electrophoresis of DNA
Evaluator: Fatma AYDINOĞLU

Agarose Gel Electrophoresis of DNA

Çağatay Utku ÇAMYAR, Tuba KENAR, Hatice ÇELİK, İrem OĞUZHAN, Deniz DURMAZ,
Hümeyra KELEŞ, İbrahim YAĞŞİ
ABSTRACT

Agarose gel electrophoresis is the most effective way of separating DNA fragments of
varying sizes ranging from 100 bp (base pairs) to 25 kb (kilo base pairs). In this experiment,
we aim to perform agarose gel electrophoresis of DNA which isolated from plant leaf
previously. While doing this experiment, the gel pouring procedure, importance of agarose gel
and the important preventions to take were observed and learned. In this experiment also the
methods to use for preparing the agarose gel, loading DNA samples and using electrophoresis
were observed and learned very well. In the end of the experiment, the results show the
separation of DNA fragments, and the discussion emphasizes the significance of this technique
in molecular biology research.

Agarose gel electrophoresis of DNA is a versatile and indispensable tool in molecular

biology, providing researchers with a simple yet powerful method for separating, visualizing,
and analysing DNA fragments in a wide range of applications. In molecular biology, while
making DNA fragment analysis, PCR and RNA analysis agarose gel electrophoresis is used.
The differences in DNA size are used while doing these analyses. This differences between their
sizes give so much information to work with.

1
INTRODUCTION

Agarose gel electrophoresis is a fundamental technique in molecular biology that

facilitates the separation and analysis of nucleic acids, primarily DNA, based on their size. This
agarose gel electrophoresis method has found widespread applications in various molecular
biology research endeavors, providing researchers with a powerful tool for visualizing and
characterizing DNA fragments [1]. The principle underlying agarose gel electrophoresis is the
migration of charged biomolecules through a porous agarose gel matrix when an electric field
is applied. DNA fragments, which are negatively charged due to their phosphate groups, migrate
towards the positive electrode and the rate of migration is influenced by the size and
conformation of the DNA fragments, allowing for their separation based on size [2]. In this
experiment, we perform the agarose gel electrophoresis of DNA that isolated from a plant, and
we estimate the sizes of DNA.

Estimating the sizes of DNA fragments in agarose gel electrophoresis is achieved by

employing a reference DNA ladder or size standard alongside the sample DNA. After loading
the DNA samples into wells and subjecting them to an electric field, smaller DNA fragments
migrate more rapidly through the agarose gel than larger ones. Following electrophoresis, the
gel is stained with a RedSafe and exposed to UV light, causing the DNA bands to fluoresce and
by comparing the migration distances of sample DNA bands to those of the known-size ladder
fragments, one can estimate the sizes of the sample DNA fragments [3]. Gel documentation
systems with dedicated software further aid in precise size determination and facilitate the
interpolation of sizes for fragments falling between ladder standards and the accuracy of these
estimations is contingent on factors such as gel concentration, electrophoresis conditions, and
the reliability of the DNA ladder utilized in the analysis [4]. So, this is how we estimate the
sizes of DNA in agarose gel electrophoresis.

By understanding the principles and nuances of this experiment, we understand the

researchers easily. With the agarose gel electrophoresis experiment, this is indispensable for a
multitude of applications, including verifying the success of PCR amplification, assessing the
purity and integrity of DNA samples, confirming the outcomes of genetic engineering
experiments, and enabling genetic analyses such as DNA fingerprinting. powerful tool for
applications ranging from assessing PCR product sizes to verifying plasmid integrity [5].

2
MATERIALS AND METHODS

The materials that used in agarose gel electrophoresis experiment are agarose powder,
TAE-TBE buffer, DNA samples, loading dye, DNA marker, an electrophoresis gel chamber,
RedSafe dye, a power supply and gel comb. The process begins with 0.8% agarose solution
which is equal to 0,24 g prepared in electrophoresis buffer. RedSafe dye is also added into the
solution to make this observation. In some experiments, Ethidium Bromide is used but, in this
experiment, RedSafe used. The reason that we use RedSafe is that it is not mutagenic as
Ethidium Bromide and that makes our experiment safer. We insert a com with wells in the gel
tray then pour the agarose solution onto the gel tray. We waited for 30 minutes to set the gel.
Then everything set to load and run the sample. In the end, the equipment for agarose gel
electrophoresis looked like as in Figure 1.

Figure 1 Agarose gel electrophoresis

In this experiment we add 1 volume of loading dye for each 5 volumes of DNA. While
loading the samples, we insert the pipet tip deep the well and load the liquid as slowly as we
can because the agarose is very sensitive and can breakable easily. After the load section, power
supply connected in, and the DNA migrate towards the anode side. Then we run the gel at a
constant voltage until the dye have migrated ¾ of the length of the gel. Then we disconnect the
power supply. After the running section completed the gel transferred to machines to give the
results. In the end the results are obtained and discussions about the experiment were made to
understand the experiment better.

3
RESULTS

Following the completion of agarose gel electrophoresis, the genomic DNA within the
gel was subjected to UV light examination. The resultant image was visualized with a help of
computer, as shown as in Figure 2.

Figure 2 The results obtained with a help of computer

Each lane displayed a unique pattern of bands, reflecting the varying sizes of DNA
fragments within the samples. The use of a DNA stain (RedSafe), enabled the visualization of
DNA bands under UV light, then see the results as nearly as expected. But in this experiment
the results that we observed was not as expected because of some mistakes that made in during
the experiment.

4
DISCUSSION

In this experiment, there are many important steps. These important steps influencing
the success of agarose gel electrophoresis include the concentration of agarose gel, the applied
voltage, and the duration of electrophoresis. In this experiment, every chemical/solution are
used to perform different functions at different stages of this experiment. For example, agarose
used for separation of DNA fragments based on their sizes. The loading dye used to add color
to the DNA samples, making them visible during loading into the gel. Each of these chemical
solutions and components contributes to the overall success of agarose gel electrophoresis by
creating an environment conducive for the separation, visualization, and analysis of nucleic
acids [6].

In this experiment, RedSafe is used instead of Ethidium Bromide. The reason of that is,
Ethidium Bromide intercalates between the base pairs of DNA, causing structural changes in
the DNA molecule. This intercalation may lead to DNA mutations during replication or repair
processes. Several studies have suggested that ethidium bromide has mutagenic potential, and
its use has raised safety considerations.

In the end of this experiment, there are some differences in the results between all the
groups. Before the experiment we expected some size but, in the end, the final sizes do not
match with the expected size. If the observed DNA bands in an agarose gel electrophoresis
experiment do not match the expected sizes, several factors could contribute to the discrepancy.
These factors can be caused by incorrect DNA marker, gel percentage, loading dye, the
electrophoresis conditions, buffer or gel pH issues. The DNA marker or size marker used may
be incorrect or degraded. The agarose gel percentage may not be suitable for the size range of
the DNA fragments. The loading dye used may interfere with DNA migration, affecting the
accuracy of size estimation. Incorrect electrophoresis conditions, such as voltage or run time,
can affect DNA migration. For the last one pH conditions in the gel or running buffer can affect
DNA migration.

5
REFERENCES

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aquaculture genetics. Aquaculture, 238(1-4), 1-37.

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migration from individual cells subjected to gel electrophoresis. Experimental cell
research, 198(2), 259-267.

[3] Holmes, D. L., & Stellwagen, N. C. (1991). Estimation of polyacrylamide gel pore size from
Ferguson plots of normal and anomalously migrating DNA fragments. I. Gels containing 3%
N, N′‐methylenebisacrylamide. Electrophoresis, 12(4), 253-263.

[4] Deagle, B. E., Tollit, D. J., Jarman, S. N., Hindell, M. A., Trites, A. W., & Gales, N. J. (2005).
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sea lions. Molecular Ecology, 14(6), 1831-1842.

[5] Zonta, E., Nizard, P., & Taly, V. (2015). Assessment of DNA integrity, applications for
cancer research. Advances in clinical chemistry, 70, 197-246.

[6] Dorfman, K. D., King, S. B., Olson, D. W., Thomas, J. D., & Tree, D. R. (2013). Beyond
gel electrophoresis: Microfluidic separations, fluorescence burst analysis, and DNA
stretching. Chemical reviews, 113(4), 2584-2667.