JSS AHER

School of Life Science

DSC 7-Instrumentation
Techniques

Topic: Agarose Gel

Electrophoresis
Sathvik B S
21L00376
BSc Biotechnology
4th Semester
Introduction
 Agarose gel electrophoresis is a widely used technique in
molecular biology for separating and analysing DNA
fragments based on their size. It is a powerful tool that
allows researchers to visualize DNA molecules, analyze
their structure, and estimate their size.
 Agarose gel electrophoresis is important in molecular
biology because it provides a simple and effective
method for separating and analysing DNA fragments. By
separating DNA fragments based on size, researchers
can gain insights into the structure and organization of
DNA and can use this information to better understand
biological processes, such as DNA replication,
transcription, and translation.

Principle of Agarose Gel

Electrophoresis
 Gel electrophoresis separates DNA fragments by size in a
solid support medium such as an agarose gel. Sample
(DNA) are pipetted into the sample wells, followed by
the application of an electric current which causes the
negatively-charged DNA to migrate (electrophorese)
towards the anodal, positive (+ve) end.
 The rate of migration is proportional to size: smaller
fragments move more quickly and wind up at the
bottom of the gel.
  DNA is visualized by including in the gel an intercalating
dye, ethidium bromide. DNA fragments take up the dye
as they migrate through the gel. Illumination with
ultraviolet light causes the intercalated dye to fluoresce.
 The larger fragments fluoresce more intensely. Although
each of the fragments of a single class of molecule is
present in equimolar proportions, the smaller fragments
include less mass of DNA, take up less dye, and therefore
fluoresce less intensely. A “ladder” set of DNA fragments
of known size can be run simultaneously and used to
estimate the sizes of the other unknown fragments

Materials and Equipment:

 Agarose
 Buffer solution (commonly used buffers include Tris-
Acetate-EDTA (TAE) and Tris-Borate-EDTA (TBE))
 DNA samples
 DNA ladder or marker (contains DNA fragments of
known sizes for comparison)
 Electrophoresis apparatus (such as a horizontal or
vertical gel electrophoresis unit)
 Power supply
 Ethidium bromide or other DNA stains
 UV transilluminator( A UV transilluminator is a device
that emits ultraviolet light and is used to visualize DNA
or protein samples that have been separated by gel
electrophoresis) or other gel imaging system
 Micropipettes and tips
Procedure
Preparing the Gel:
1. Measure out the desired amount of agarose powder and
add it to a flask containing the appropriate volume of
buffer solution. The recommended agarose
concentration depends on the size range of the DNA
fragments to be separated and can range from 0.5% to
3%.
2. Heat the mixture in a microwave or on a hot plate until
the agarose is completely dissolved. Allow the mixture
to cool slightly before pouring it into the gel casting tray.
3. Place the comb into the gel casting tray to create wells
for sample loading. Allow the agarose to solidify at room
temperature for about 30 minutes.
Loading the Samples:
1. Mix the DNA sample with a loading buffer, which
contains dyes to visualize the sample during loading and
to monitor the electrophoresis process. The most
commonly used loading buffer is 6x Loading Dye.
2. Carefully remove the comb from the gel casting tray to
avoid disrupting the wells
3. Load the DNA ladder or marker in the first well, followed
by the DNA samples in the remaining wells. Load equal
amounts of DNA in each well to ensure an accurate
comparison of band intensities.
Running the Gel:
1. Place the gel into the electrophoresis apparatus and fill
the apparatus with enough buffer solution to cover the
gel.
2. Connect the electrodes to the power supply, ensuring
that the positive electrode is connected to the end of
the gel with the wells and the negative electrode is
connected to the opposite end of the gel.
3. Apply an electric current to the gel. The optimal voltage
and running time depend on the size of the DNA
fragments to be separated and the agarose
concentration of the gel. For example, a 1% agarose gel
run at 100 volts for 45 minutes can separate DNA
fragments ranging from 200 bp to 10,000 bp.
4. Monitor the electrophoresis process, making sure that
the gel is running smoothly and that the bands are not
smearing. If necessary, adjust the voltage or running
time

Staining the Sample:

1. Staining is a crucial step in agarose gel electrophoresis
that makes DNA visible. The most commonly used DNA
stains are ethidium bromide and SYBR Green, which
intercalate between the DNA bases and fluoresce under
UV light.
 2. Staining solutions are typically poured over the gel or
the gel is submerged in the staining solution. The
staining solution must be carefully prepared and
handled to ensure that the DNA is not damaged or
degraded.

Visualization:
1. After staining, the gel is placed under UV light in a gel
documentation system or transilluminator. The DNA
fragments appear as bands that can be visualized and
photographed.
2. It's important to wear protective glasses and handle the
stained gel carefully to avoid exposure to UV light, which
can be harmful to both the eyes and the DNA.
3. Staining can also affect the downstream applications of
the DNA, such as cloning or sequencing. For this reason,
it's important to choose a staining method that is
compatible with the intended application and to
carefully control the staining conditions to avoid
potential interference.
Factors Affecting Electrophoresis

Gel Concentration: The concentration of the agarose gel

can affect the resolution of the DNA fragments, with higher
concentrations resulting in tighter separation of small
fragments but the reduced separation of larger fragments.
Voltage and Run Time: The voltage applied to the gel and
the duration of the run can also affect the resolution of the
DNA fragments, with higher voltages and longer run times
allowing for greater separation of the fragments.
Buffer Composition: The buffer solution used in the
electrophoresis chamber can affect the migration rate of the
DNA fragments, with certain buffers being more suitable for
specific applications.
Temperature: The temperature of the buffer solution and
the gel can also affect the migration rate of the DNA
fragments, with higher temperatures resulting in faster
migration rates.
 These factors should be optimized for each experiment
to achieve optimal separation and resolution of DNA
fragments for different applications.
Applications of Agarose Gel
electrophoresis
some of the most common applications of agarose gel
electrophoresis:

1. DNA fragment analysis: Agarose gel electrophoresis is

commonly used to separate DNA fragments of different
sizes. This can be used to analyze PCR products,
restriction digests, and other DNA samples.
Advantages:
 Simple and inexpensive technique
 Can analyze large quantities of DNA simultaneously
 Can be used to analyze various types of DNA fragments
Limitations:
 Cannot distinguish between DNA fragments of similar
sizes
 Cannot provide information about the sequence or
identity of the DNA fragments
 Smaller fragments may be difficult to visualize on the gel
2. RNA analysis: Agarose gel electrophoresis can also be
used to separate and analyze RNA fragments, such as
those obtained from RT-PCR or Northern blotting.
Advantages:
 Allows for the detection and quantification of RNA
molecules
 Can be used to analyze different types of RNA (e.g.
mRNA, rRNA, tRNA)
Limitations:
 RNA can be more fragile and susceptible to degradation
compared to DNA, which can affect the accuracy of the
results
 RNA fragments may be more difficult to separate and
visualize compared to DNA fragments

3. Protein analysis: Agarose gel electrophoresis can also

be used to separate and analyze proteins. This is often
done using a technique called native polyacrylamide gel
electrophoresis (PAGE), which separates proteins based
on their size and charge.
Advantages:
 Can separate and analyze proteins based on size and
charge
 Can be used to detect and quantify specific proteins
 Can be used to compare protein expression levels in
different samples
Limitations:
 Requires specific stains and techniques to visualize the
proteins on the gel
 Cannot provide information about the identity or
function of the proteins
 May not be suitable for analyzing large or complex
proteins

4. Southern blotting: Southern blotting is a technique

used to detect specific DNA sequences in a sample. This
involves separating the DNA fragments on an agarose
gel, transferring them to a membrane, and then
hybridizing the membrane with a labelled DNA probe
that is complementary to the target sequence.
Advantages:
 Allows for the detection and quantification of specific
DNA sequences
 Can be used to analyze different types of DNA samples
(e.g. genomic DNA, plasmid DNA)
Limitations:
 Requires additional steps (e.g. DNA transfer, probe
hybridization) that can be time-consuming and
technically challenging
 May be less sensitive compared to other techniques
such as PCR or next-generation sequencing

5. DNA sequencing: Agarose gel electrophoresis is often

used as a preparatory step in DNA sequencing. This
involves separating the DNA fragments on an agarose
gel, excising the bands of interest, and then purifying the
DNA fragments for use in sequencing reactions.
Advantages:
 Allows for the determination of the sequence of DNA
fragments
 Can be used to analyze different types of DNA samples
(e.g. genomic DNA, PCR products)
Limitations:
 Requires additional steps (e.g. gel excision, DNA
purification) that can be time-consuming and technically
challenging
 May not be suitable for analyzing large or complex
genomes

6. Genetic mapping: Agarose gel electrophoresis can also

be used to create genetic maps of organisms by
separating DNA fragments that contain genetic markers.
Advantages:
 Can be used to construct genetic maps of organisms
  Can be used to identify and analyze genetic markers
Limitations:
 May require additional steps (e.g. PCR amplification) to
obtain the DNA fragments for analysis
 May not provide information about the function or
phenotype of the genetic markers

Troubleshooting and optimization

Troubleshooting and optimization in agarose gel
electrophoresis can help to improve the accuracy and
reliability of the results. Here are some key points to consider
for troubleshooting and optimization:
1. Agarose concentration: The concentration of agarose in
the gel can affect the resolution and separation of DNA
fragments. Higher concentrations of agarose can
improve the resolution of small fragments but may make
it more difficult to separate larger fragments. Lower
concentrations of agarose can improve the separation of
larger fragments but may result in decreased resolution
for smaller fragments.
2. Running buffer: The pH and composition of the running
buffer can also affect the separation and resolution of
DNA fragments. It is important to ensure that the
running buffer is at the appropriate pH and contains the
necessary ions for optimal electrophoresis.
3. Voltage and run time: The voltage and run time can
affect the migration of DNA fragments on the gel. Higher
voltages and longer run times can improve the
separation of DNA fragments, but may also increase the
risk of overheating and damage to the gel.
4. DNA loading: Overloading or underloading the gel can
affect the resolution and separation of DNA fragments. It
is important to ensure that the appropriate amount of
DNA is loaded onto the gel and that the loading buffer is
compatible with the gel and running buffer.
5. Staining and visualization: The choice of stain and
visualization method can affect the sensitivity and
accuracy of the results. Some stains may be more
sensitive to certain types of DNA fragments, and
different visualization methods may be more
appropriate for different applications.
6. Gel quality: The quality of the gel can also affect the
accuracy and reliability of the results. It is important to
ensure that the gel is free from defects (e.g. bubbles,
cracks) that may affect the migration of DNA fragments.
Troubleshooting: If problems arise during agarose gel
electrophoresis, it is important to troubleshoot and
identify the source of the problem. Common issues include
uneven migration of DNA fragments, smearing, and poor
resolution. Troubleshooting may involve adjusting the
concentration of agarose or running buffer, optimizing the
voltage and run time, or changing the DNA loading
conditions.
Overall, troubleshooting and optimization in agarose gel
electrophoresis can help to improve the accuracy and
reliability of the results. By carefully considering factors such
as agarose concentration, running buffer, voltage and run
time, DNA loading, staining and visualization, gel quality, and
troubleshooting, researchers can ensure that they obtain
accurate and meaningful results from their experiments.

Variations of Agarose Gel

Electrophoresis
Agarose gel electrophoresis is a widely used technique for
separating and analyzing biomolecules, such as DNA, RNA,
and proteins, based on their size and charge. There are
several types of agarose gel electrophoresis techniques used
in various applications. Here are some of them:
1. Standard agarose gel electrophoresis: This is the most
common type of agarose gel electrophoresis used for
separating DNA fragments of different sizes. The agarose
gel is made by dissolving agarose powder in a buffer
solution and then pouring it into a gel tray. The DNA
sample is loaded into wells made in the gel, and an
electric current is applied to separate the DNA
fragments based on their size.
2. Pulse field gel electrophoresis (PFGE): This technique is
used for separating large DNA fragments, such as those
found in bacterial and fungal chromosomes. PFGE uses a
 specialized gel matrix and an alternating electric field to
produce a unique banding pattern that can be used to
identify different strains of bacteria or fungi.
3. Two-dimensional gel electrophoresis (2DGE): This
technique is used for separating and analyzing proteins
based on their size and charge. 2DGE involves two
separate steps: isoelectric focusing (IEF) and SDS-PAGE.
IEF separates proteins based on their isoelectric points
(pI), while SDS-PAGE separates them based on their
molecular weight.
4. Reverse transcription PCR (RT-PCR) and gel
electrophoresis: This technique is used to detect RNA
expression levels. The RNA is first reverse-transcribed
into cDNA using a reverse transcriptase enzyme. The
cDNA is then amplified using PCR, and the resulting
products are separated on an agarose gel to detect the
expression levels of specific genes.
5. Denaturing gradient gel electrophoresis (DGGE): This
technique is used for analyzing DNA sequence variation.
DGGE uses a gradient of denaturants (such as urea or
formamide) to create a temperature gradient in the gel.
This allows DNA fragments of similar sizes but different
sequences to be separated based on their melting
behaviour.
6. Single-strand conformation polymorphism (SSCP): This
technique is used for detecting single-base mutations in
DNA. SSCP involves denaturing the double-stranded
DNA into single-stranded DNA and then separating the
 resulting strands on an agarose gel based on their
conformational changes.

Advancements in Agarose Gel

Electrophoresis:

some current research advancements in agarose gel

electrophoresis:
1. High-resolution agarose gels: Scientists are developing
high-resolution agarose gels that can separate DNA
fragments with higher accuracy and resolution. These
gels have a higher concentration of agarose, which
reduces the pore size and allows for the separation of
smaller DNA fragments. Additionally, the use of
advanced imaging techniques, such as fluorescent dyes
and confocal microscopy, allows for better visualization
of the separated DNA fragments.
2. Digital agarose gel electrophoresis: This new technique
combines agarose gel electrophoresis with digital PCR
(dPCR) technology. The DNA sample is first loaded onto
a microfluidic chip and partitioned into thousands of
individual droplets. Each droplet undergoes PCR
amplification, and the resulting products are then
loaded onto an agarose gel for separation and analysis.
Digital agarose gel electrophoresis allows for more
 accurate quantification of DNA fragments and reduces
the risk of false positives.
3. Automated agarose gel electrophoresis: Automation is
becoming increasingly popular in agarose gel
electrophoresis. Scientists are developing automated
systems that can load and run gels, as well as analyze
and interpret the resulting data. This reduces the risk of
human error and allows for higher throughput.
4. Development of new dyes and stains: Scientists are
developing new dyes and stains that can improve the
resolution and sensitivity of agarose gel electrophoresis.
For example, the use of ethidium homodimer-2 (EthD-2)
dye has been shown to improve the detection of low-
concentration DNA samples.
5. Portable agarose gel electrophoresis systems:
Researchers are developing portable agarose gel
electrophoresis systems that can be used in the field for
rapid and accurate detection of pathogens. These
systems are designed to be lightweight, battery-
powered, and easy to use, making them ideal for point-
of-care testing in remote or resource-limited settings.
Overall, these advancements in agarose gel electrophoresis
technology are improving the accuracy, sensitivity, and speed
of DNA analysis, and are likely to have a significant impact on
research and diagnostics in various fields, including genomics,
microbiology, and clinical medicine.
Conclusion
 Agarose gel electrophoresis is a widely used technique
for separating and analyzing biomolecules, such as DNA,
RNA, and proteins, based on their size and charge.
 There are several types of agarose gel electrophoresis
techniques used in various applications, such as
standard gel electrophoresis, pulse field gel
electrophoresis, 2D gel electrophoresis, RT-PCR and gel
electrophoresis, DGGE, and SSCP.
 Current research in agarose gel electrophoresis is
focused on developing high-resolution gels, combining
agarose gel electrophoresis with digital PCR technology,
automating the process, developing new dyes and
stains, and creating portable systems for point-of-care
testing.
 These advancements in agarose gel electrophoresis
technology have significant implications for research and
diagnostics in various fields, including genomics,
microbiology, and clinical medicine.
 Agarose gel electrophoresis remains an important tool in
molecular biology, and future advances in the field will
likely continue to improve the accuracy, sensitivity, and
speed of DNA analysis.
Bibliography
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