Gel Electrophoresis Lab Report

Stephen Price

July 8, 2018

Biology
Abstract:

This experiment is about gel electrophoresis. Gel electrophoresis is one of the most common
technique for separating macromolecules by size or charge, and is widely used in biochemical
research and pharmacology. The gel experiment was proceeded by loading dye samples into the
prepared gel and electrophoresed by the power supply. The results were ponceau G and xylene
cyanol for the unknown 1, bromphenol blue and ponceau G for unknown number 2, and the
mixture of xylene cyanol and pyronin Y for unknown number 3. Besides, the farthest dye sample
was pyronin Y. Overall, the experiment went well because all the data were achieved properly.
In addition, there was no sign of errors in the gel electrophoresis experiment

Introduction and Background:

Gel electrophoresis is one of the most common technique for separating macromolecules by size
or charge, and is widely used in biochemical research and pharmacology. Macromolecules, such
as DNA, is often used for CSI or criminal cases to identify fingerprints. To examine, a sample of
DNA is placed in an agarose gel, and an electrical charge is sent through by the power supply,
based on size. The negatively charged molecules of DNA moves toward to another the positively
charged molecules. After separating, the bands are stained with dyes, such as methylene blue or
ethidium bromide in order to be seen. The result of DNA is a series of bands with different sizes.
The farthest band from the start of gel is the smallest band of DNA. Meanwhile, the closet band
to the start of gel is the largest band of DNA.

Figure 1: Example of band of DNA.

Agarose is a popular gel that often used in chemistry labs and other science fields. Agarose gel is
formed by mixing agarose with the buffer, and allows time to become a gel matrix. By adjusting
the concentration of agarose used in the gel, the sizes can be determined. Specifically, the more
concentrated the gel, the longer DNA takes to move. Conversely, the less concentrated of the gel,
the fast the DNA moves. There are many advantages of using agarose gel. One of the main
advantages is that agarose is a nontoxic gel, that does not harm to human. Besides, agarose can
recovers samples by melting the gel, digesting with enzyme agarose or treating with salts.
Objective & Hypothesis:

To find out whether restriction enzymes, cut down the DNA strand into different sizes of the
fragments. The restriction enzymes are chemical which cuts the DNA strand into fragments in a
palindromic DNA sequence which might be of different sizes.

Lytic enzyme as a protein derivative can be used in gel electrophoresis to facilitate DNA
breaking. Restriction enzymes when introduced in DNA strand use the gel electrophoresis
process to separate the fragments.

Materials & Method:

The dye was loaded on the samples to be separated. Power was turned on and the approved
voltage set, after which a running buffer was added to cover the gel. The gel box leads were
attached to the power supply ensured they were workingproperly. The DNA samples were
loaded and the lid was replaced to the gel. The power was turned on and gel ran until the dye was
separated to different distances.

The main materials used in the gel electrophoresis lab were 1.6 percent of agarose mixture, 1X
TBE, unknowns, dye samples and power supply. The experiment began by casting the agarose
gel with 1X TBE and dissolved in a hot bath. Then, the mixture was filled, and covered about
half of the height of the comb teeth of the casting tray. The process to condense took around 15
minutes. Once the agarose was solidified, the comb was removed and ready to use.

Before loading the dye samples into the gel, the gel was filled with 1X TBE buffer that just
covered the surface of the gel. The given dye samples, including three unknowns, were loaded
into the gel by using the digital pipettor. To load the first dye sample, the pipette tip was used to
draw 10 micro liter of the dye. Using the dominant hand, the pipette was steadily poured into the
well above the buffer layer from left to right. Besides, the non-dominant hand was used to guide
the pipet through the surface of the buffer and positioned directly over the well. The dye was
sunk to the bottom of the well because the dye was already mixed with surcrose to increase the
density. The experiment continued processing for the rest of the samples.

Once all of the dye samples were loaded, the lid was placed on the electrophoresis chamber, and
the electrical cords were connected to the power supply. 100 Volts was used for the whole
process. When the power supply was turned on, the gel ran slowly until the dye in lane 3 was 0.5
cm from the end of the gel. Later, the result was all recorded and analyzed.

Data:

Results:
The first suspect as the control gap to ensure that molecules were working correctly. The DNA
proteins and RNA were separated at 100 mV gel electrophoresis.

Table 1: Results of the Gel Electrophoresis

Lane Sample Number of bands (+) or (-) Migration

distance (cm)
1 Bromphenol blue 1
2 Methyl orange 1
3 Pronceau G 1
4 Xylene cyanol 1
5 Pyronin Y 1
6 Unknown #1 2
7 Unknown #2 2
8 Unknown #3 2

After the results, the unknown number 1 was identified as a mixture of xylene cyanol and
ponceau G; bromphenol blue and ponceau G for unknown number 2; and the mixture of xylene
cyanol and pyronin Y for unknown number 3. From observing, the farthest dye molecule through
the gel was pyronin Y because this type of gel was the smallest from total of eight dye samples.
The smaller the gel is, the faster the sample goes. Conversely, the bigger the gel is, the slower the
sample goes. Besides, the charges of the dye samples were also determined. Negative charge was
found for bromphenol blue, methyl orange, pronceau G, xylene cyanol, and unknown number 2.
Positive charge was found for pyronin Y. The only mixture of negative charge and positive
charge was unknown number 3. When doing electrophoresis, the mixture of unknown 3 went
two ways, including xylene cyanol and pyronin Y. In the aspect, the result seemed to be correct
because pyronin Y was positive, and xylene cyanol was negative; therefore, the mixture had to
go two different directions.

Conclusion:

The results of this lab proved that my hypothesis was correct: each restriction enzyme did
create a different DNA fingerprint for lambda DNA. Restriction enzymes do cut DNA into a
variety of sizes because the certain sequence that the enzyme is looking for could appear any
number of times, and it does not have to be perfectly spaced out. The restriction enzymes will cut
DNA in different places regardless of how many times the required sequence appears.

The shorter DNA fragments did move farther through the gel than bigger pieces. The
amount that the fragments move is based on length. The smaller DNA fragments are easier to
move through the gel than longer pieces. The spaces between the bands in the DNA fingerprint
prove that the DNA was cut into varying sizes because some move more than others.

There were many sources of error that were present when we did this lab. The linear
equation from the logarithmic graph (F2) was not completely accurate because the best fit line
did not touch every single point on the scatter plot. Our teacher gave us the actual base pair
values so we could see the difference. Another error was that we did not have access to a
micropipette and we had to use less accurate pipettes. They were not very effective and caused
some of the lambda DNA and restriction enzyme mixtures to miss the wells in the gel and get
mixed into the buffer.

Another big error was that the gels were supposed to be run through the electrical current
for about an hour and a half. We ran out of time and only ran the gels for twenty minutes. We
also stained them for too long after we took them out of the electrophoresis box. We had no UV
light, which was needed to properly see the banded pattern for the type of dye we used. All of
these factors contributed to why no DNA fingerprint was present on our gel.

According to the results, the hypothesis was correct as the DNA fragments were separated to
different distances regarding their sizes. This data supported the hypothesis as DNA in the gel we
moved from negative to the positively charged anode at different distances. The shorter ones
moved for a wrong distance than the longer ones.

From the experiment, I acquired the knowledge that DNA strands can be cut into a different size
which can be separated according to their sizes. This can be used in real life situation by
separating DNA fragments in order to clone any specific band.

The results completed as a group in class didn't come out as expected. This is because the gel
required to be run for 30-35 minutes but time was not enough to allow this. Also, apparatus use
was inappropriate since we used a pipette instead of a micro-pipette. The control experiment was
the DNA sample which was placed in a test tube with water while the experimental group was
the DNA sample placed in the gel.