Presented By:

Sathvik B S
21L00376
BSc Biotechnology
4th Semester
AGAROSE GEL
ELECTROPHORESIS

Presented By:
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.
INTRODUCTION
 PRINCIPLE
● DNA fragments are separated based on their size as they move through an
agarose gel matrix towards a positive electrode. Smaller fragments move
faster through the gel than larger fragments, separating the fragments
based on size.

● The separation is achieved by applying an electric field across the gel,

causing the negatively charged DNA fragments to move towards the
positive electrode. The gel matrix acts as a sieve, slowing down the
movement of larger fragments more than smaller fragments, resulting in
the separation of the fragments based on size.
PRINCIPLE
MATERIALS AND EQUIPMENTS
AGAROSE
Agarose is a type of polysaccharide
derived from seaweed that is used
to create a gel matrix for DNA
electrophoresis.
BUFFER
SOLUTION
commonly used buffers include
Tris-Acetate-EDTA (TAE) and Tris-
Borate-EDTA (TBE))
DNA Ladder or
Marker
contains DNA fragments of known
sizes for comparison
Electrophoresis
apparatus
Parts of apparatus like
electrophoresis chamber, gel boat,
power supply and electrodes
UV Transilluminator
A UV transilluminator is a device
that is used to visualize DNA or
protein samples that have been
separated by gel electrophoresis
DNA Dye
Dyes like ethidium bromide are
used to stain the DNA for
illumination
1)Preparing the gel

2)Loading the sample

3)Running the sample

4)visualization
 1) Preparing the gel
● Adding the required amount of agarose powder to the
appropriate volume of buffer solution
● Recommended amount of agarose depends on the size range
of the DNA which can range from 0.5% to 3%
● Heat the mixture until the agarose is completely dissolved,
then allow the mixture to cool
● Add the DNA dye ethidium bromide to it before pouring it into
the gel carefully remove the comb from the gel casting tray to
avoid disrupting the wells casting tray
● Place the comb into the casting tray to create wells for sample
loading
● Allow the agarose to solidify at room temperature for about
30 minutes
 2)Loading the sample
● Mix the DNA sample with a loading buffer, The most commonly
used loading buffer is 6x loading buffer
● Carefully remove the comb from the gel casting tray to create
wells without disrupting it
● Load the DNA ladder or marker in the first well, followed by the
DNA sample in the remaining wells, ensure to load equal
amounts of DNA sample
 3)Running the gel
● Place the gel into the electrophoresis chamber and fill the
chamber with enough buffer solution to cover the gel
● Connect the electrodes to the power supply, ensure that the
positive electrode is connected to the end of the gel with the
walls and the negative electrode is connected to the opposite
end of the gel
● Monitor the electrophoresis process making sure the gel is
running smoothly
 4)Visualization
● Then the gel is placed under UV light in a transilluminator. The
DNA bands fragments appear as bands due to their size
difference
● The dye like ethidium bromide helps us to visualize the DNA
under UV light
● Staining can affect the cloning and sequencing, so its important
to choose a staining method that is compatible with intended
application
● Its important wear protective glasses and handle the stained gel
carefully to avoid exposure to UV light which harmful for our
eyes and DNA
 APPLICATIONS
DNA fragment analysis
• Agarose gel electrophoresis is commonly used to separate DNA fragments of different
sizes
• This can be used to analyse various DNA samples
RNA analysis
• Agarose gel electrophoresis can also be used to separate and analyse RNA fragments,
such as those obtained from RT-PCR or northern bloating
Protein Analysis
• It is used to separate and analyse proteins.
• It is often done using a technique called PAGE(polyacrylamide gel electrophoresis
Southern Blotting
• It is used to detect specific DNA sequences in a sample
• This involves separating the DNA fragments and transferring them to a membrane and then
hybridizing it with a DNA probe that is complementary to the target sequence
DNA Sequencing
• It is often used as a preparatory step in DNA sequencing
• This involves separating the DNA fragments, excising the band of interest and then
purifying the DNA fragments for sequencing
 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:
• 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: 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.
 TROUBLESHOOTING AND OPTIMIZATION
4. DNA loading: 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: 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.
 CONCLUSION
● Agarose gel electrophoresis is a fundamental technique in molecular
biology research, allowing researchers to separate and analyze DNA
fragments based on their size.

● It is a powerful tool for identifying genetic variations, analyzing gene

expression, and studying DNA-protein interactions.

● The technique is widely used in many areas of research, including

genetics, forensics, and medical diagnostics.

● Understanding the principles and procedures of agarose gel

electrophoresis is essential for anyone working in molecular biology,
and it remains one of the most widely used techniques in the field .
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