Name : Paloma V.
Gomez Date : December 14, 2024
Group # 1 Offer Code: 6034
LABORATORY EXPERIMENT 7
GEL ELECTROPHORESIS
I. INTRODUCTION
Gel electrophoresis is a widely used laboratory technique for the separation and
analysis of biomolecules, such as DNA, RNA, and proteins. It employs an agarose gel,
which contains a network of tiny pores, to distinguish molecules based on their size and
electrical charge. By applying an electric current, charged biomolecules are driven
through the gel towards electrodes with opposite charges. Due to their uniform negative
charge, DNA fragments consistently migrate toward the positive electrode, with smaller
fragments move faster through the gel's pores compared to larger ones.
To aid in the process, loading dye is typically added to the samples. This dye not only
provides a visual cue for monitoring the progress of the gel run but also increases the
density of the sample, ensuring it sinks neatly into the wells of the gel. This simple yet
powerful technique serves as a cornerstone in molecular biology, enabling researchers to
study and manipulate genetic material with precision.
Objectives :
This laboratory experiment aims to;
1. To demonstrate how to set up an electrophoresis box;
2. To use properly a micropipette in transferring liquid to the gel in the
electrophoresis box;
3. To separate molecular fragments based on size and charge using gel
electrophoresis;
4. To interpret the banding pattern you obtain after gel electrophoresis
5. To communicate the observations and results based on the simulation in a
laboratory report;
6. To appreciate the use of technology, such as simulated lab experiments, to learn
laboratory techniques and processes.
II. MATERIALS
A. APPARATUS
P2 Pipette P2 Tip Box
P20 Pipette P20 Tip Box
P200 Pipette P200 Tip Box
P1000 Pipette P1000 Tip Box
Agarose Gel Power Supply
Gel Electrophoresis Box Power Supply Leads
Microcentrifuge Trash
B. CHEMICAL REAGENTS
S1 Solution S3 Solution
S2 Solution 1x Sodium Borate Buffer
III. PROCEDURE
A. Prepare the Electrophoresis Box.
1. Select the agarose gel.
2. Place the agarose gel into the electrophoresis box.
3. Orient the wells of the gel towards the negative electrode.
4. Pour the flask with the 1x Sodium Borate Buffer over the gel box.
B. Centrifuge the Solution Tubes.
1. Select the microcentrifuge to open the lid.
2. Select and move each of the solution tubes to the microcentrifuge.
3. Balance the microcentrifuge by placing the tubes so filled receptacles
are symmetrical.
4. Select the microcentrifuge to close the lid.
5. Press the pulse button to briefly spin the liquid to the bottom of the
solution tubes.
6. Select the microcentrifuge to open the lid.
7. Remove the tubes and put them back on the rack.
8. Select the microcentrifuge to close the lid.
C. Draw Up Solution 1.
1. Select the P20 micropipette.
2. Select the volume setting to set the volume to 10μl and then select Save
volume.
3. Select the P20 tip box to open it.
4. Move the P20 micropipette to the P20 tip box to attach a tip.
5. Select the P20 tip box to close it.
6. Select the S1 solution tube to open it and place the micropipette into it.
7. Draw up solution 1 by pressing down the plunger until it reaches the first
stop and releasing the plunger slowly.
8. Close the S1 solution tube.
D. Pipette Solution 1 into Well 1.
1. Select the P20 micropipette and move it over the gel electrophoresis box
to prepare to load the gel.
2. Move the micropipette to load the sample into the first well by selecting
lane 1.
3. Dispense solution 1 by pressing down on the plunger until it reaches the
first stop.
4. Move the micropipette over the trash and press down on the eject button to
eject the tip.
E. Pipette other Solutions into the Remaining Wells.
1. Pipette solution 2 into lane 2 following the same process as before.
2. Pipette solution 3 into lane 3 following the same process as before.
3. Remember to make notes in your lab notebook! Click the pencil to record
the lanes into which you pipetted each solution. This will allow you to
interpret your results. You can drag a pipette with a tip over the open gel
box to see the gel content list again if you have closed the dispense
solution dialog.
Take Note: The convention when loading a gel is to load it from left to right.
Lane 1 : Solution 1
Lane 2 : Solution 2
Lane 3 : Solution 3
F. Conduct Gel Electrophoresis.
1. Place the cover over the electrophoresis box.
2. Select the red and black cables and connect them to the red and black
connection points on the power supply, and the red and black connection
points on the lid of the gel electrophoresis box.
3. Turn on the power supply.
4. Set the voltage to 130V.
5. Set a timer to 10 minutes.
6. Start the timer on the power supply.
7. Wait until the timer has elapsed.
8. Turn off the power switch.
9. Unplug the electrodes from both the power supply and the gel box.
10. Carefully remove the cover from the electrophoresis box to inspect your
gel.
IV. DATA AND RESULTS
The results of the experiment closely matched my predictions, and I was able to
observe how the principles of gel electrophoresis played out in practice. The blue band
migrated the least distance, the purple band moved farther, and the yellow band traveled
the farthest, just as I anticipated. This pattern highlights how gel electrophoresis separates
molecules based on size and charge, with smaller molecules like the yellow dye moving
more quickly and covering greater distances through the gel matrix, while larger
molecules like the blue dye lag behind. The purple band behaved mostly as expected but
showed slight overlap with the yellow band. I think this could be due to minor variations
in the experiment, such as the gel’s composition or loading inconsistencies, or possibly
because the molecular properties of the purple and yellow dyes are more similar than I
initially thought. Even with these minor discrepancies, the results validated the
predictions and reinforced my understanding of how molecular size and charge influence
migration in a gel. It was satisfying to see these principles come to life, as the experiment
demonstrated the effectiveness of gel electrophoresis as a tool for molecular separation.
RESULT FEEDBACK
I think the greatest impact on my results came from the balance of charge and size
for each dye molecule. While size mattered, the charge-to-mass ratio was the key factor
in how far the dyes migrated. For example, the purple dye, though larger, moved farther
because it had a higher negative charge per mass. I also believe the accuracy in preparing
and loading the gel played a significant role. Proper sample loading, consistent gel
composition, and maintaining the correct voltage were crucial for clear separation. Any
mistakes here could have altered the results, so attention to detail was important for
getting the expected outcomes.
REFLECTION
V. QUESTION AND ANSWER
1. Why is it important to orient the gel with the wells nearest to the negative electrode
when running DNA or negatively charge protein molecules ?
When running DNA or negatively charged protein molecules in gel electrophoresis, it is
vital to position the gel with the wells nearest to the negative electrode (cathode). This
ensures that the negatively charged molecules migrate toward the positive electrode
(anode) under the applied electric field. If the gel is oriented incorrectly, the samples will
travel in the wrong direction, potentially leaving the gel entirely and resulting in sample
loss and failed analysis.
2. What will happen if you have air bubbles in your tip before loading the samples into
the wells?
The presence of air bubbles in the micropipette tip during sample loading can disrupt the
process and compromise results. Air bubbles may prevent the sample from being
dispensed properly into the wells, leading to incomplete loading or splashing, which can
contaminate adjacent wells. Furthermore, inconsistent sample volumes caused by air
bubbles can result in uneven band intensities during analysis, making it crucial to verify
that the tip is bubble-free before loading.
3. In using the micropipette, why is it necessary to press the pipette’s plunger up to the
first stop only? Explain
Proper use of a micropipette requires pressing the plunger only to the first stop to ensure
accurate measurement and aspiration of the intended liquid volume. Pressing beyond the
first stop while aspirating can cause excessive liquid to be drawn up or introduce air
bubbles, reducing accuracy. During dispensing, the first stop releases the measured
volume, and the second stop ensures complete expulsion of any residual liquid. This
method guarantees precise and consistent handling of samples, which is essential for
reliable results.
VI. CONCLUSION
VII. REFERENCES
Fats and oils: Emulsification. Institute of Food Science and Technology. (2018, June 25).
https://www.ifst.org/lovefoodlovescience/resources/fats-and-oils-emulsification
Khan Academy. (2023). Emulsions. Khan Academy.
https://www.khanacademy.org/science/chemistry/acids-bases-and-ph/properties-of-acids-and-bas
es/a/emulsions
Khan Academy. (2023). Surfactants and Emulsions. Khan Academy.
https://www.khanacademy.org/science/chemistry/acids-bases-and-ph/properties-of-acids-and-bas
es/a/surfactants-and-emulsions
Leal-Calderon, F. (2012). Emulsified lipids: formulation and control of end-use properties. OCL,
19(2), 111–119. https://doi.org/10.1051/ocl.2012.0438
LibreTexts. (2023). Lipid Tests. LibreTexts.
https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Book%3A_Organic_Chemistry_wit
h_a_Biological_Emphasis_(Soderberg)/14%3A_Lipids/14.01%3A_Reactions_and_Tests_of_Lip
ids
National Institutes of Health. (2023). Lipid Metabolism. National Institutes of Health.
https://www.nih.gov/health-information/lipid-metabolism
ScienceDirect. (2023). Micelles. ScienceDirect.
https://www.sciencedirect.com/topics/chemistry/micelles
VIII. REFLECTIONS
1. Before performing the experiment, indicate your confidence level in demonstrating the
skills stated in the objectives by putting a check mark [a] corresponding to your
confidence level using the rating scale below.
1 2 3 4 5
OBJECTIVE very unconfident slightly neutral confident very
unconfident enough confident
Before doing the simulation, I believe I can ….
a. Demonstrate how
to set up an
electrophoresis box
b. Use properly a
micropipette in
transferring liquid
to the gel in the
electrophoresis box
c. Separate molecular
fragments based on
size and charge
using gel
electrophoresis
d. Interpret the
banding pattern
obtained after gel
electrophoresis
e. Communicate the
observations and
results of the
simulation
experiment in the
laboratory report
2. What difficulties, if any, did you encounter in performing the simulation for gel
electrophoresis? List and describe these difficulties. How did you overcome these
difficulties?
3. Answer the following question;
(a.) Do you appreciate the use of simulation to learn the technique of gel electrophoresis?
Specify which feature of the simulation you appreciate. You may state more than 1
feature.
(b.) Which feature of the simulation do you think needs improvement? Why?
4. How many times did you need to navigate the simulation until you successfully oerform
gel electrophoresis properly and without mistakes? How long, in minutes, did it take you
to perform gel electrophoresis with mastery?
5. Which part of this experiment can you apply the following:
(a.) Critical Thinking
(b.) Communication
(c.) Creativity
(d.) Collaboration
6. Would you recommend the use of simulation for some laboratory experiments? If yes,
indicate some situations in which you could use simulations in replacement for actual
laboratory experiments? If no, state your reasons.
7. After performing the simulation, reevaluate your confidence level based on the objectives
of the experiment.
1 2 3 4 5
OBJECTIVE very unconfident slightly neutral confident very
unconfident enough confident
Before doing the simulation, I believe I can ….
f. Demonstrate how
to set up an
electrophoresis box
g. Use properly a
micropipette in
transferring liquid
to the gel in the
electrophoresis box
h. Separate molecular
fragments based on
size and charge
using gel
electrophoresis
i. Interpret the
banding pattern
obtained after gel
electrophoresis
j. Communicate the
observations and
results of the
simulation
experiment in the
laboratory report
