Acıbadem Mehmet Ali Aydınlar University

MBG109
General Biology I
Lab Report
Faculty Arts and Sciences
Department Molecular Biology and Genetics
Course Instructor Dr. Perinur Bozaykut Eker
Lab No 4-5
Experiment Name DNA Isolation & Agarose Gel Preparation and Electrophoresis
Experiment Date 30.11.2022 - 14.12.2022
Due Date 21.12.2022
Submitted By Ceylin Baykoç
Student ID 221403022
To Mail ceylin.baykoc@live.acibadem.edu.tr
Submitted To Erdoğan Oğuzhan Akyıldız
Signature
Purpose of Experiment:

DNA extraction and agarose gel electrophoresis are the basic techniques to obtain good-quality DNA in sufficient quantity. In
DNA isolation, saliva is obtained to get genomic DNA. Later, this DNA solution has mixed with several chemicals (Detergent,
Saline Solution and Cold Absolute Ethanol) to separate DNA from other proteins and organic compounds chemically. Then this
solution has centrifuged to make a separation of the DNA from dissolved salts and sugars. After the centrifuge process DNA
proteins were released from the solution completely. With Ethanol, impurities from the DNA have totally removed and
eventually pure DNA has been made.

In agarose gel electrophoresis, the main purpose is making a confirmation of DNA and analyzing the rate of migration across
the agarose gel with electrophoresis results. Based on this, the size of the DNA in means of base pairs can be found
approximately. Therefore, the quantity and the quality of DNA can be visualized on electrophoresis. This visualization can occur
with staining chemicals which are Tibo Gold and Bromophenol Blue. This process happens with these staining chemicals and 1X
TAE Buffer to understand the size of the genomic DNA sample by comparing it to the DNA ladder which shows the base pair
numbers with fluorescent signals with bands.

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Materials:

For DNA Isolation: For Agarose Gel Electrophoresis:

• Pasteur pipette • Equipment and • Transilluminator

• 30% Saline Solution (0.9% NaCl)
Reagents 
 • Agarose 

• Cold Absolute Ethanol • Pipetman
• Submarine gel • Tibo Gold 

• Deionized water (ddH2O) • Pipette tips apparatus 
 • 1X TAE Buffer
• Eppendorf tubes (1.5 ml) • Water bath • Power supply 
 • Loading Dye
• Detergent • Erlenmeyer 
 (Bromophenol Blue)

• Falcon Tubes (50 mL) • Microwave 
 • Parafilm 

• Gloves • Pipetman and pipette • DNA Ladder 

tips • Gloves

Methods:

• For DNA Isolation:

1. 4 ml of tap water is used to rinse the mouth to obtain saliva.

2. 50 mL falcon tube is filled with saliva solution which is obtained from the first step.
3. 5 mL of Saline solution (0.9% NaCl) has added to saliva solution.
4. 1 mL of detergent has added to solution which is obtained from the previous step.
5. After adding detergent to the solution, the solution has blended with micropipette for 3 minutes without causing foam.
6. 5 mL of ice-cold absolute Ethanol (96%) has added into solution carefully.
7. After waiting for at least one minute without interrupting, DNA precipitation and phases have been visualized.
8. The DNA has transferred to 1.5 mL Eppendorf tube using micropipette (P1000).
9. The DNA has centrifuged at 10.000 rpm for 1 minute.
10. The supernatant has discarded, and the DNA pellet has air dried.
11. 200 𝜇𝐿 Double distilled water (ddH2O) has added to the DNA.
12. The DNA has incubated at 56°C for 60 minutes and stored at -20°C.

• For Agarose Gel Electrophoresis:

1. 0.3 g of agarose has weighed for 1% mini gel into an Erlenmeyer flask.
2. Agarose has boiled with 30 mL of 1X TAE (Tris-acetate-EDTA) in microwave until the gel is homogenously transparent.
3. The electrophoresis tray has set, combs and stoppers has put.
4. The agarose gel has poured and waited for polymerization for 20 minutes.
5. 1X TAE has poured onto agarose gel to cover the surface.
6. The stoppers have taken out from the agarose gel.
7. 1X TAE has added to electrophoresis tank to make agarose gel submarine.
8. 1 𝜇𝐿 of loading dye (Bromophenol Blue) has put on a piece of parafilm.
9. 10 𝜇𝐿 of DNA sample has putted and mixed with loading dye via micropipette on a piece of parafilm.
10. 1 𝜇𝐿 of Tibo Gold has added to solution and mixed with micropipette.
11. The samples and ladder have loaded to the wells.
12. The agarose gel has run at 120V for 40 minutes.
13. The agarose gel has analyzed under UV trans illuminator, and the results are recorded.

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Results:

For DNA isolation, when a new chemical is added to the DNA solution every step has observed and recorded the final
appearance of the solution. When saline solution and detergent have added to the DNA solution, visually there was no change
recorded. However, when cold absolute Ethanol (96%) has been added to the DNA solution the DNA can be seen as a new
phase. Then, this DNA phase has transferred into an Eppendorf tube for centrifuge at 10.000 rpm for one minute. After
discarding the supernatant and adding double distilled water to the solution the isolation has done for agarose gel
electrophoresis.
For agarose gel electrophoresis, a sufficient amount of agarose gel has prepared which is 1 gram to 100 mL of water then boiled
in a microwave until it is homogeneously blended. Then electrophoresis tray has prepared with a comb and stoppers. In this
step 1X TAE has put onto the gel and onto the electrophoresis tray. The sample has been prepared with several chemicals which
are Bromophenol Blue and Tibo Gold and loaded into agarose gel wells. In this experiment, the well has selected 5th from the
left of the 2nd row. After that, the gel has run at 120V for 45 minutes. Lastly, the gel was exposed to UV light and the picture
was taken with a gel documentation system. According to the results of the UV Transilluminator, the treadmill of the DNA
sample and the fluorescent signal has been observed and a band has seen on the gel. However, the DNA band has seen smeared.
Lastly, in this experiment red color has appeared at the beginning of the well.

Figure 1.1: The gel electrophoresis results Figure 1.2: In this figure the fifth well has shown
which taken from UV Transilluminator. above which was selected for this experiment.

Discussion:

In DNA isolation; to obtain pure DNA without contaminants, physical and chemical methods are used. Firstly, the Saline solution
has used to help neutralize the DNA charge and make the DNA less hydrophilic, which is why it becomes less soluble in water.
The salt also keeps the proteins dissolved in the water and helps in the removal of proteins that are attached to DNA. Secondly,
detergent is used to break down the phospholipid bilayer of the cell and release the DNA into the solution. Lastly, cold absolute
ethanol has used in this experiment to make hydrogen bonds with the water molecules located around DNA to precipitate DNA.
In this experiment, ethanol has added to the solution ice-cold temperature. Ice-cold ethanol caused the DNA not to denature.
In agarose gel electrophoresis, DNA fragments are separated using electrophoresis based on how big they are in comparison. A
block of agarose gel containing DNA fragments is loaded which is called a ladder, and the gel is then placed in a chamber with
a suitable liquid buffer solution which is TAE (Tris-acetate-EDTA). Since in this experiment low current (120V) has used TAE has
been selected instead of TBE (Tris-Boric acid-EDTA). Also, 1X TAE is selected because it is used for a long run of DNA. Since
genomic DNA is used in this experiment 1X TAE is suitable for this condition. In this experiment, TAE is used to maintain ion
balance for electrical current. Since DNA is negatively charged, it will be attracted toward the positive pole in an electric field.
Visualizing DNA cannot be possible without a stain and loading dye, therefore in this experiment, Tibo Gold has been used as a
stain to detect the approximate number of nucleotides in that DNA. Tibo Gold is an intercalation agent that sticks to DNA’s
double strand. Loading dye which is Bromophenol Blue makes the DNA sample denser which causes it to sink into the agarose
gel's wells and helps in the tracking of DNA movement. In UV Transilluminator the results have seen therefore the DNA band
has seen smeared, that problem can be based on the salt concentration of solution and nuclease contamination. If the salt
concentration is too much this problem may occur. The red color which appeared at the beginning of the well means that the
DNA sample has remained in the well.

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