Agarose Gel

Lab Report 3
Maria Kamal Katha
ID:22336048
Submitted for: BTE258

Biotechnology Lab III
Section:05
Submitted to: MD Tawsif Ur Rashid

Brac University
Summer Semester 2024
Experiment No:03 Date:23.06.2024
Name of The Experiment: Agarose gel electrophoresis of DNA
Objective: This experiment aims to determine the presence or absence of PC products and
quantify the product's size (length of the DNA molecule).
Principle: Gel electrophoresis is a basic biotechnology technique that separates macromolecules

according to their size and charge. It is frequently used to analyze and manipulate DNA, RNA, or
protein samples. DNA has a net negative charge that is proportional to its size. When placed in an
agarose matrix and exposed to an electric field, DNA molecules will move toward the positive
electrode. The agarose gel is made up of tiny holes. Smaller molecules move faster than larger
molecules because they produce less friction in the agarose matrix. In gel electrophoresis, samples
to be separated are applied to a porous gel medium made of a material such as agarose.
Agarose separates DNA based on size or charge. Agarose creates a gel-like network and creates
pores to move DNA with these pores’ DNA travels. As much as the agarose percentage is low
DNA can move easily in the gel. Agarose is a purified form of agar, a gelatinous substance
extracted from red algae. Agarose gels are made by first adding powdered agarose to a liquid buffer
and boiling the mixture until the agarose dissolves. This molten agarose is then cooled to about
40-60°C, poured into a gel mold called a casting tray, and allowed to solidify. Before solidification
occurs, a comb is placed in the casting tray to create a row of wells into which samples are loaded
once the comb is removed from the solidified gel. During electrophoresis, the gel is submersed in
a chamber containing a buffer solution and a positive and negative electrode. The DNA to be
analyzed is forced through the pores of the gel by the electrical current. Under an electrical field,
DNA will move to the positive electrode (red) and away from the negative electrode (black).
Several factors influence how fast the DNA moves, including; the strength of the electrical field,
the concentration of agarose in the gel, and most importantly, the size of the DNA molecules.
Smaller DNA molecules move through the agarose faster than larger molecules. DNA itself is not
visible within an agarose gel. The DNA will be visualized by the use of a dye that binds to DNA.
Gels with lower agarose concentrations are used to separate larger DNA fragments. A 1% agarose
gel will resolve duplex DNA chains ranging from -600-20,000 bp. A loading dye is mixed with a
DNA sample before getting electrophoresis in a 1:4 ratio. The function of the loading dye is to add
density to the DNA and to track its movement along the gel. Loading dye can be of 2 types:
• Upper dye: Xylene cyanol for 10,000-40,000 bp
• Lower dye: Bromophenol blue for 400-500 bp
Loading dye has 30%glycerol which adds weight or density to the DNA. With 1% agarose TBE/
TAE buffer (50ml) is added to balance the pH. To visualize DNA in UV we need to use Ethidium
bromide (EtBr).
Apparatus and Reagents:

Apparatus
1. 500 ml flask
2. Water bath
3. Electrophoresis chamber
4. Power supply
5. Gel casting tray and combs
6. Staining tray
7. Gloves
8. Pipette and tips
Reagents
1. Agarose
2. ТВЕ Buffer
3. TE buffer
4. Loading dye
5. Ethidium bromide (EtBr)
6. DNA sample
TBE Buffer Preparation:
1. 10.8 g of Tris is dissolved in 900 ml of distilled water.
2. 5.5 g of Boric acid is dissolved in the same solution.
3. 4 ml of 0.5 M Na2EDTA (pH 8.0) is added.
4. The volume is adjusted to 1 Liter.
5. The solution is stored at room temperature.
Loading Dye 1
Reagents:
1. 0.25% Bromophenol blue
2. 0.25% Xylene cyanine
3. 30% glycerol
Loading Dye 1
Reagents:
1. 0.125% Xylene cyanine
2. 0.125% Bromophenol blue
3. 0.1% SDS (For density)
4. 40% sucrose (For density)
Procedure:
Preparation of agarose gel
1. 0.4 g of agarose powder is measured and added to a 100 ml flask.
2. 50 ml of TBE Buffer is added to the flask. (The total gel volume will vary depending on
the size of the casting tray.)
3. The agarose is melted in a microwave until the solution becomes clear.
Note: If a microwave is used, the solution is heated for several short intervals. The solution
should not be allowed to boil for long periods as it may boil out of the flask.
4. The solution is allowed to cool to about 50-55°C, with the flask occasionally swirled to
cool evenly.
5. The combs are placed in the gel casting tray.
6. The melted agarose solution is poured into the casting tray and allowed to cool until it
becomes solid.
7. The combs are carefully pulled out and the tape is removed.
8. The gel is placed in the electrophoresis chamber.
9. Enough TAE Buffer is added so that there is about 2-3 mm of buffer over the gel.
Note: Gels can be made several days before use and sealed in plastic wrap (with combs).
If the gel becomes excessively dry, it is allowed to rehydrate in the buffer within the gel
box for a few minutes before loading samples.
Loading sample
1. The DNA sample is mixed with an equal volume of ethanol and centrifuged at 14,000 rpm
for 10 minutes.
2. The ethanol in the supernatant is discarded and the pellet is air-dried.
3. The pellet is dissolved in 20 ul TE buffer (10 ul more can be added if needed).
4. 10 ul of the DNA sample is mixed with 5 ul of loading dye.
5. The order each sample will be loaded on the gel is recorded, including who prepared the
sample, the DNA template, what organism the DNA came from, controls, and the ladder.
6. Each sample is carefully pipetted into separate wells in the gel.
7. 10 ul of the DNA ladder standard is pipetted into at least one well of each row on the gel.
Note: If multiple gels are being run, the DNA ladder is loaded in different lanes on each
gel to avoid later confusion.
Running the gel
1. The lid is placed on the gel box, connecting the electrodes.
2. The electrode wires are connected to the power supply, ensuring the positive (red) and
negative (black) are correctly connected. ("Run to Red" is remembered.)
3. The power supply is turned on to about 100 volts. The maximum allowed voltage varies
depending on the size of the electrophoresis chamber and should not exceed 5 volts/cm
between electrodes.
4. The current is checked by looking for bubbles forming on each electrode.
5. The direction of the current is checked by observing the movement of the blue loading dye,
which will take a couple of minutes. (It will run in the same direction as the DNA.)
6. The power is left running until the blue dye approaches the end of the gel (30 minutes).
7. The power is turned off.
8. The wires are disconnected from the power supply.
9. The lid of the electrophoresis chamber is removed.
10. Using gloves, the tray and gel are carefully removed.
Gel Staining
1. Using gloves, the gel is removed from the casting tray and placed into the staining (EtBr)
dish.
2. The gel is allowed to stain for at least 25-30 minutes (the entire gel will become dark blue).
3. The stain is poured off (the stain can be saved for future use).
4. The gel and staining tray are rinsed with water to remove residual stains.
5. The tray is filled with warm tap water (50-55°C). The water is changed several times as it
turns blue. Gradually, the gel will become lighter, leaving only dark blue DNA bands. The
gel is destined completely overnight for best results.
6. The banding pattern of DNA resolved through the gel is recorded by photography.
Photographs of ethidium-bromide-stained gels may be made using transmitted or incident
UV light.
7. The data is recorded while the gel is fresh, as very light bands may be difficult to see with
time.
Note: Gels stained with blue stains are stable for long periods. When destining is complete,
the gel is removed from the water and allowed to dehydrate. Dark bands can be seen in a
dried gel for weeks or months.
(a)
Result
Band
(b)
Figure 3: (b)Agarose gel under UV light is compared with (a) 1 Kb ladder
Result:
No DNA band was seen.
Discussion:
We have used genomic DNA which contains all the genes. Genomic DNA is typically very large,
often exceeding 12 kb in length. For this experiment, a 1 kb DNA ladder is used with a maximum
size of 12 kb as a reference. Instead of large fragments, we observed fragments below 250 base
pairs (bp), with a prominent band around 200 bp. This discrepancy suggests that the DNA isolation
process may not have been successful.
The cetyltrimethylammonium bromide (CTAB) method is a common technique for isolating DNA.
It involves several steps, including cell lysis, removal of proteins and polysaccharides, and
precipitation of DNA. If the pellet did not contain DNA, it suggests that the isolation steps were
not properly executed. This could be due to inadequate cell lysis, improper precipitation, or loss
of DNA during the washing steps. During the CTAB isolation process, RNase is often used to
degrade RNA. Since RNase was not used in your process, the observed band around 200 bp could
be RNA, which was not degraded and thus appeared on the gel. RNA molecules are typically
smaller than genomic DNA, which aligns with the observed fragment sizes.
Conducting the isolation process outside of a laminar flow hood can lead to contamination from
environmental sources. Contaminants can interfere with the isolation process and result in poor
quality or absence of genomic DNA. Dust, airborne microbes, and other contaminants can degrade
or interfere with the DNA isolation.
The gel running time is 35 minutes, whereas it is recommended to run it for 45-60 minutes.
Running the gel for a shorter time can result in insufficient separation of DNA fragments, making
it difficult to visualize larger fragments. A longer run time allows larger DNA fragments to migrate
further through the gel matrix, providing better resolution and clearer bands.
Ensure all steps of the CTAB isolation process are followed meticulously, including proper cell
lysis, DNA precipitation, and washing steps. Include an RNase treatment step to degrade RNA and
prevent it from appearing on the gel. Perform the isolation process in a laminar flow hood to
minimize contamination. Run the gel for the recommended duration (45-60 minutes) to achieve
better separation and visualization of DNA fragments.
Precaution:
1. Care should be taken to avoid contact EtBr with skin.

2. Melted agarose should be poured into one corner of the casting tray to avoid the formation
of bubbles.

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Lab Report 3

Maria Kamal Katha

Submitted for: BTE258

Submitted to: MD Tawsif Ur Rashid

Name of The Experiment: Agarose gel electrophoresis of DNA

Principle: Gel electrophoresis is a basic biotechnology technique that separates macromolecules

Apparatus and Reagents:

No DNA band was seen.

1. Care should be taken to avoid contact EtBr with skin.

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