FACULTY OF APPLIED SCIENCES

BACHELOR OF SCIENCE (HONS.) BIOLOGY

BIO615
MOLECULAR BIOLOGY

LAB REPORT
AS2015B1

TITLE: GEL ELECTROPHORESIS

PREPARED BY:
1. AMIRA NASIRAH BINTI JEMAT (2020986757)
2. ALYA ZAHIDAH BINTI ZULKIFLI (2020106337)
3. DAYANG NOR SHAFIQAH BINTI AWANG SHUKERAN (2020994777)
4. SITI NURSUHADAH BINTI JANTAN (2020327101)

PREPARED FOR: TS. DR. ASLIZAH MOHD ARIS

SUBMISSION DATE: 18/7/2022
INTRODUCTION

Gel electrophoresis is used to separate DNA fragments based on the differences in

their sizes. Gel quantitation is a technique used by research scientists to get an estimation of
the size and quantity of DNA fragments in an agarose gel. The technique involves the
comparison of a known quantity of DNA to an unknown quantity of DNA on an Agarose gel.
The gel is stained with loading dye followed by ethidium bromide (EtBr) followed and
photographed. The brightness of a particular fragment, as well as the distance traveled, can
be used to estimate the quantity and size of a particular fragment.

Gel electrophoresis sorts DNA fragments in order of size by using electricity run
through a gel matrix. DNA is made into different sizes through restrictive enzymes, enzymes
that cut DNA into smaller pieces so that they can be analyzed. Once the DNA is cut, it is
stained then inserted into the wells. When the electricity flows, the DNA is pulled from the
negative side to the positive side because DNA has a negative charge. Smaller molecules
move more easily while larger molecules move more slowly through the gel. This leads to
smaller molecules and therefore molecules that have been cut by restriction enzymes will
travel further toward the negative pole. This lab needed to test out this process and see if it
was successful. Since this has been done so many times by thousands of scientists, the
hypothesis is that this experiment will be able to separate DNA fragments through DNA gel
electrophoresis.

OBJECTIVE

● To be an efficient and effective way of separating nucleic acids (to separate DNA
fragments).
● To allow agarose's high gel strength for the handling of low percentage gels for the
separation of large DNA fragments.
MATERIALS AND APPARATUS

Agarose, protein sample, comb, tank and running model, power supply, analytical balance,
beaker, casting frame, casting chamber, pipette, TAE buffer, spatula, UV light transilluminator,
chiller, bunsen burner.

PROCEDURE

1. 1g of agarose was weighed by using analytical balance.

2. The agarose was mixed and poured into the 100mL 1xTAE buffer.
3. The mixture was boiled until clear in colour. The mixture was stirred continuously to
fasten the process.
4. 5μL of gel stain was added into the mixture. The mixture was carefully mixed until the
stain was fully dissolved.
5. The casting chamber was prepared which served as the mold.
6. Then, the gel was poured carefully into the casting tray.
7. The comb was placed across the end of the casting tray to form wells when the gel
solution solidified.
8. The gel was placed in the chiller until it solidified.
9. After it solidified, the comb was removed and the electrophoresis set was prepared.
10. Ladders were placed for size referencing and the gel was put into the chamber.
11. 1xTAE buffer solution was poured into the casting chamber until the gel was fully
covered.
12. The sample solutions were added to each well approximately 10μL. The pipette tip
was ensured to be inserted deeply enough into the well to minimize spillage into the
surrounding area.
13. The casting tank was covered once all the samples had been loaded.
14. The electrodes were ensured to be in the right position and connected to the power
supply.
15. The voltage was set correctly to75-90V.
16. Any movement of the bands were observed and the run was stopped once the bands
had nearly reached the end of the gel.
17. The DNA bands were visualized using UV light transilluminator and the bands were
compared with the DNA ladder.
RESULT

Figure 1: Agarose gel under the UV light.

The result above shown that the band for three of the sample is at 2.0 kb (2000 base pair).
DISCUSSION

Gel electrophoresis is a method for separating DNA fragments or other

macromolecules like RNA and proteins based on their size and charge. There are three major
steps done to perform gel electrophoresis. First is preparation of agarose gel, second is the
placement of agarose gel and the sample in the gel box and lastly is screening the agarose
gel under UV light.

From the result above, it showed that three of the sample is on the middle part of the
gel with the quantity of 2 kb (2000 base pair). The DNA fragment is in the center part of the
agarose gel might be because the DNA fragment is not to short and not too long. Shorter
fragments of DNA will move quicker through the pores of the gel matrix as the gel runs. After
running the gel for a period, the shortest bits of DNA will be towards the positive end of the
gel, while the longest strands of DNA will be near the wells.

As simple as it seems, there are also challenges need to be overcome when

performing gel electrophoresis. Due to the highly dynamic liquid, when doing gel
electrophoresis, the result could be affected including the quality of image under the ultraviolet,
recycling of DNA as well as protein examination. These problems occur is due to several
factors.

The first factor that causes the result to failed is pH condition. When electricity passes
through the liquid, a redox reaction occurs at two electrodes, producing hydrogen ions and
hydroxide ions. However, because gel electrophoresis is normally done at pH 8.3, this
circumstance might substantially alter the pH condition of electrophoresis. Changes in pH
might make the experiment impossible to carry out.

The second factor that causes the result cannot be observe is due to the difficulties of
the molecule to move because they have multiple shape. The channel form of agarose gel for
electrophoresis is fixed. DNA and protein molecules, on the other hand, have a range of
appearances. As a result, not all molecules can flow through the channel readily. In some
circumstances, study utilizes this approach to isolate molecules with distinct structures.
However, the gels have several limits. Linearly branched polymers, for example, prefer
channel structures, whereas globular molecules flow more effectively in Brownian rectifiers.
To improve the effectiveness of gel electrophoresis, the difficulties created by channel shapes
must be addressed.
 The third component is the distortion of molecules produced by the strength of the
electric field. Clearly, DNA molecules and proteins have polar structure produced by hydrogen
bonds and ionic bonds. However, the strength of the electric field is a critical component in
weakening those bonds or possibly destroying the entire structure, preventing us from getting
exact data.

To overcome this problem, there are a few steps need to be done. The pH condition is
the first difficulty that must be solved to improve the quality of the electrophoresis result.
Changes in pH make long-duration electrophoresis impractical. As a result, the buffer's
resilience should be highlighted and enhanced. To allow the largest sample size, the fraction
of weak-based buffer should be tuned.

Another factor to consider is the very specific form of the gel's channels. To increase
electrophoresis effectiveness, a more flexible gel should be designed to meet the demands of
multiple combination samples at the same time. In terms of ionic bond damage and medium
entanglement, the assisted reagent may be used to avoid deformation by providing a stable
ionic bond structure and measurement range throughout the experiment.

More studies are required to determine the best electrophoresis conditions, including
gel thickness, electric intensity, and temperature, in order to validate the optimal electric field
intensity.
CONCLUSION

Electrophoresis gel, in conclusion, is an analytical technique used to separate nucleic acid

molecules (DNA or RNA) based on size. It is used to investigate the lengths of DNA and how
they relate to various strands. This method is efficient for separating molecules up to 20 kb in
size. In forensics, it is also utilized to determine personal identity. Furthermore, we can
conclude from these findings that agarose gel electrophoresis can be an efficient and
successful method of separating nucleic acids. By carrying out this experiment, we were able
to get a better understanding of the role of restriction enzymes and agarose gel
electrophoresis play in the size and cutting of DNA.

REFERENCE

Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H., (2012). Agarose gel electrophoresis for
the separation of DNA fragments. J Vis, 62(3923). doi: 10.3791/3923. PMID: 22546956;
PMCID: PMC4846332.