PH161.

1 Laboratory Exercise 11 WORKSHEET

Electrophoresis

Name: Joshua Pionelo Section: 1 Date submitted: April 24, 2021

I.What is Gel Electrophoresis?

1. Prepare an agarose gel to check your PCR products with an expected size of 800bp and
1.2kb. How much grams of agarose would you need to prepare a 50ml gel?

Based on the table for recommended agarose gel concentration for DNA separation,
the weight per volume concentration required to check PCR products with an expected size of
800bp and 1.2kb is 0.7%. We will use this value to create an agarose gel solution of 50 mL.

Given:
w/v (%) = 0.7 % or 0.007
Volume of Solution = 50 mL
Required
Mass of solute = ?
𝑤 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 (𝑔)
(%) = × 100
𝑣 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 (𝑚𝐿 )
𝑥
0.7% = × 100
50 𝑚𝐿

𝑥
0.7% 50 𝑚𝐿 × 100
( = )
100 100
 𝑥
0.7% 50 𝑚𝐿 × 100
( = )
100 100
𝑥𝑔
(0.007 = )
50 𝑚𝐿
𝑥𝑔
50𝑚𝐿 ቀ0.007 = ቁ 50𝑚𝐿
50 𝑚𝐿

50𝑚𝐿 × 0.007 = 𝑥𝑔

0.35𝑔 = 𝑥𝑔 𝑠𝑜𝑙𝑢𝑡𝑒

The total amount of solute needed to create a 50 mL agarose gel solution with a
0.7% weight per volume concentration is 0.35 g. This agarose gel solution will be used to
separate DNA products with 800bp and 1.2kb.

2. How are buffers used in electrophoresis?

Buffers are used in electrophoresis as a medium to transmit charge. Buffer also maintains
the gel at a stable pH, minimizing changes that could occur in the protein or nucleic acid if
subjected to unstable pH (Collins, 2018).

II. Simulation: Running a gel

Which level did you select? 3
A. Results
1. Compare your results with your ideal results by making a note and describing any
differences you observe between the results. Explain why the bands ended up in those
relative positions. Is this what you predicted would happen?
 Upon the conclusion of the simulation, the actual results of the experiment matched the
ideal result. Based on the actual results, the blue band, which is perceived to be in the middle of
the ranking on which is the largest in size, travelled the least among the three dyes. The purple,
the largest among the three, travelled father than the blue band and the yellow band, the one with
the smallest size, travelled the farthest from the starting point.
If we observe the ideal and actual result, we may notice that the colors of the bands are
more faded than the bands in the ideal result. The size of the bands is also different between the
two. The bands in the actual result were larger than in the ideal result.
Before the simulation began, the principle of the experiment was discussed briefly. It was
discussed that smaller molecules will move through the pores in the gel faster than larger molecules.
The simulation also discussed that among the three dyes, the yellow one is the smallest, followed
by the blue band, and followed by the purple band, which is the largest among the bands.
The details mentioned were used to come up with a predicted result showed in the picture.
However, there was a noticeable difference between the ideal and actual results. The student
predicted that the purple, which is the largest, would not travel farther than the blue band, and the
yellow band, being the smallest would be the one to travel the farthest. The predicted result for the
yellow band matches the result of both the ideal and actual results. However, for the purple and
blue bands, the result was different.
Based on the results, it can be inferred that there are factors other than the molecular size that
influenced the rate of diffusion of the molecules in the agarose gel. If we are testing solely for the
nucleic acid, the predicted results would be the same with the ideal and actual results, since the
migration flow of nucleic acids would solely depend on the molecular weight (Yilmaz, Gok, &
Ozic, 2012). The sample used in the simulation was different types of dye as mentioned in the
context portion of the simulation. At the end of the simulation, it was discussed that another
variable might influenced the rate of migration. Since the process of gel electrophoresis uses
electrical charge to separate molecules, the charge of the molecule can influence its rate of
migration.
 2. What part of the procedure do you think had the greatest impact on the results you
obtained?

There are a number of variables that influence the rate at which a molecule migrates in
the agarose gel through electrophoresis. The viscosity and pore size of the support media or gel
affects the rate of migration. A higher viscosity slows down the rate of migration and a greater
pore size speed up the migration.
Increasing the strength of the electrical field by raising the voltage and increasing the
temperature used for the electrophoresis will increase the mobility and rate of migration.
In the simulation, the agarose gel was already prepared in advance, hence there was no
manipulation happened in the viscosity and pore size of the support media. The same is true
with the strength of the electrical field as the voltage used was only 130 V.
Since there were no other set-ups in terms of medium viscosity and power of the
electrical field and the rate or speed of migration was not observed quantitatively, I may infer
that the power of the electric field have played the greatest impact in the result since the
substance used is greatly influenced by the charge of the electric field as stated in the
discussion at the results part of the simulation.

B. Reflections
(paste your answers to the questions here)
III. Application of gel electrophoresis: verifying a recombinant plasmid by gel
electrophoresis
A. Results
1. Compare your results with your ideal results by making a note and describing any
differences you observe between the results. Explain why the bands ended up in those
relative positions. Is this what you predicted would happen?

Getting directly to the point, the ideal, actual, and predicted results are far different from
each other. One of the major difference among the three is the distance of the gap of each bands.
In the predicted results, the gap between each bands are almost identical and each bands are not
that far from each other. For both the actual and ideal results, the distance between bands are
varied, some are too far apart and some are closer to each other.
Plasmid DNA can exist in three conformations – supercoiled, circular (OC), and linear.
These different plasmid conformation influences the migration rate in a gel media. As we can
observe in the different results of each sample, supercoiled plasmid DNA travelled further than
most of the samples. This is because a small, compact supercoiled knot of ccc-DNA sustains less
friction against the agarose matrix than does a large, floppy open circle of oc-DNA. Nicked
plasmid DNA is a plasmid with one DNA strand cut or “nicked”; this releases the supercoiling and
leaves a large, floppy circle with slow mobility in agarose. Therefore, for the same over-all size,
supercoiled DNA runs faster than open-circular DNA.
Results for gK -
gK+
gA –
gA+
gLIG
 2. What part of the procedure do you think had the greatest impact on the results you
obtained?

All procedures used in the experiment have a significant impact on the results obtained.
We learned that the factors that influence the rate of migration are molecular size of the sample,
agar gel medium, and power of the electric field. In the current experiment, we cannot deny the
effects of all variables, however, in my own conclusion, the variable or the procedure that gave
significant impacts on the result is the sample used during the experiment.
The different conformation of the plasmid DNA, as well as the number of base pairs (bp)
gave different results during the experiment. Since DNA has an overall negative charge, we may
only use the molecular size of the sample in determining which molecule would travel the farthest
and the fastest among the samples. In the virtual experiment, it can be observed that supercoiled
plasmid DNA reached the farthest distance among the different samples, since it has a smaller size,
thus having less friction from the agar gel media.
B. Reflections
(paste your answers to the questions here)