Laboratory Report #9 – ELECTROPHORESIS

Name:
Text Maria Amor Macapallag
Abstract:
Gel electrophoresis is a laboratory technique for separating DNA, RNA, and protein
mixtures based on their molecular size. An electrical field pushes the molecules to be separated
through a gel with microscopic holes in gel electrophoresis. The molecules move at a rate that is
inversely proportional to their lengths through the pores in the gel. This implies that a little DNA
molecule will move further across the gel than a larger DNA molecule. In addition, gels for DNA
separation are frequently constructed of agarose, a polymer that comes in powdered flakes.
When agarose is heated and allowed to cool in a buffer, it forms a solid, somewhat spongy gel.
The gel is a matrix of agarose molecules bound together by hydrogen bonding and forming small
pores at the molecular level. In this activity, different sets of samples are given which also shows
different types of error when performing an electrophoresis. Furthermore, in visualizing a result,
the DNA fragments glow when a gel is stained with a DNA-binding dye and exposed to UV
light, allowing us to view the DNA present at various positions along the length of the gel. A
band is a well-defined "line" of DNA on a gel. Each band comprises a significant number of
DNA fragments of the same size that have all gone to the same location as a group. On a gel, a
single DNA fragment (or even a small collection of DNA fragments) would not be seen.
Introduction:
According to a study, Gel electrophoresis is a typical molecular biology laboratory
technique for identifying, quantifying, and purifying nucleic acids. The approach is frequently
used for nucleic acid isolation and analysis because to its speed, simplicity, and adaptability.
Nucleic acids in the range of 0.1–25 kbp may be separated for examination using gel
electrophoresis in a matter of minutes to hours, and separated nucleic acids can be retrieved from
the gels with reasonably high purity and efficiency. Additionally, electrophoresis was first used
to separate nucleic acids in the early 1960s. Nucleic acids were frequently segregated at the time
using density gradient centrifugation based on sedimentation velocities, which are governed by
nucleic acid size and conformation. Density gradient centrifugation took a long time, required
heavy equipment, and required a large number of samples. Moreover, gel electrophoresis has
become an ubiquitous approach for separating nucleic acids in molecular biology. This analytical
and preparative approach is not only essential in typical workflows such as molecular cloning
and PCR, but it also plays a critical role in nucleic acid separation and analysis in upcoming
technologies such as genome editing and next-generation sequencing.
Methodology:
Gel electrophoresis is a technique that allows scientists to separate charged molecules
according to size. This includes DNA, RNA and proteins. To conduct electrophoresis, the gel
must first be prepared. This may be done in practically any laboratory; agarose gels are the most
commonly used. To produce the gel, agarose powder is combined with an electrophoresis buffer.
This combination is then heated until the agarose has been completely dissolved and
incorporated into the buffer solution. To summarize, the method for electrophoresis are the
following:
1. Prepare agarose gel
2. Pour into casting tray with comb and allow to solidify
3. Add running buffer, load samples and marker
4. Run gel at constant voltage until band separation occurs
5. View DNA on UV light box and show results

In this experiment, the materials, reagent and equipment used in both Protocols are:
1. Agarose powder (Low EEO)
2. Baker’s Paper cup
3. Electrophoresis machine Microwave oven
4. Gel Loading buffer or DNA loading Dye
5. GelRed dye
6. 0.5 X TBE buffer (running Buffer)

The gel is often constructed of agarose, a polysaccharide that forms a semi-solid, somewhat
porous gel when heated in a buffer solution. The gel generates small indentations called wells at
one end, where the researcher deposits the DNA samples under examination, as well as reference
samples of known length, known as a DNA ladder. Moreover, when the gel is placed in a
conducting solution and a voltage is given, the fragments begin to migrate through the gel, the
smaller ones first, followed by the bigger, slower ones. They gradually coalesce into spectrum-
like bands based on size. Finally, when this happens, the researcher switches off the power, fills
the gel with a DVA-binding dye, and analyzes the specimens under UV light. Using the ladder as
a guide, the researcher may calculate the size of each piece in a visible band. Individual DNA
pieces are too tiny to view, therefore only bands are seen.
Results & Discussion:
Electrophoresis is most commonly employed in molecular biology as a method for DNA
analysis, but it is also utilized in forensics to identify materials from a crime scene. In order to
obtain reliable findings, it is critical that the sources of error in this approach be reduced.
Contamination of the DNA sample is one source of inaccuracy. If there is foreign DNA in the
sample, the gel will have more bands than a gel with only the pure material. In electrophoresis, a
variety of distinct components are at action, and each one is crucial in identifying the type of
molecules being investigated. Scientists can identify what sort of molecules they are looking at
based on how fast they move, how strong the electrical current is, the precise properties of the
gel, the shape of the molecules, the size of the molecules, the temperature of the solution, and
other parameters. In addition, to keep the molecules in place, they are dyed in varied striations
throughout the gels, creating the appearance of a series of colorful bands. This is one of the most
significant procedures in DNA analysis because it allows scientists to extract DNA proteins and
analyze them closely to identify their unique properties.
Identify the following on the gel electrophoresis sample given

Agarose gel Identify the approximate Describe the trouble

amount of the visible shooting problem from
band the gel
 The first (1) sample
shows an approximate
amount of 400 bp. The
second (2) sample shows
an approximate amount of
about 500 bp. Lastly, the
third (3) sample shows a
large amount of about an
approximately of 500bp
and higher.
1 2 3 ladder
The visible bands in well
1 are approximately
235bp, 500bp, 765bp, and
1135bp in size, according
to the gel electrophoresis
data. The visible bands in
well 2 are around 865bp,
1135bp, and 2245bp in
size. The observable
bands in well 3 are 865bp
and 1135bp in size. The
visible band sizes for well
4 are around 1135bp,
Dna 1450bp, and 2245bp.
Ladder
1 2 3 4
The “thickness” of the
band correlates directly
with the amount of DNA
present. Since the shown
band is as thicker than
the bands in the ladder, it
is likely that the sample
simply contains more
DNA than the ladder.
Uneven heat distribution
during electrophoresis
can lead samples in the
warmer center of the gel
to move faster than ones
at the cooler margins.
The uneven heat
distribution causes a
"smile" effect, making it
difficult to compare
samples put in various
sections of the gel.
 1-4 sample Band smearing and
The visible band size in streaking are cause by:
well 1 is approximately
625bp, 1885bp, and • Salt
2885bp, according to the concentration of
data. The visible band in the sample is too
well 2 is approximately high
625bp, 2885bp, and
2885bp in size. 3 and 4 • Excessive power
are the same. However, and heating
there are weak bands in
this gel between 625bp • The sample
and 2885bp, above spilled out of well
2885bp and below 625bp.
• Incomplete
digest nuclease
contamination.

Dna
Ladder
1 2 3 4

References:
• Khan Academy, (n.d.). Gel Electrophoresis. Retrieved from:
https://www.khanacademy.org/science/ap-biology/gene-expression-and-
regulation/biotechnology/a/gel-electrophoresis
• Scitable by Nature Education. (n.d.). Gel Electrophoresis. Retrieved from:
https://www.nature.com/scitable/definition/gel-electrophoresis-286/
• Thermo Fisher Scientific, (n.d.). Nucleic Acid Gel Electrophoresis—A Brief Overview and History.
Retrieved from: https://www.thermofisher.com/ph/en/home/life-science/cloning/cloning-
learning-center/invitrogen-school-of-molecular-biology/na-electrophoresis-education/na-
separation-overview.html
• Pasquesi, Andy. (July, 2018). How Is DNA Visualized Using Gel Electrophoresis?. Retrieved from:
https://sciencing.com/dna-visualized-using-gel-electrophoresis-5516941.html