Names: ___________________________________Pd: ______ Color group: ____________________
Agarose Gel Electrophoresis with Food Color
Agarose gel electrophoresis can resolve molecules based on charge, size, and shape. In this laboratory you
will use gel electrophoresis to separate molecules present in different food color mixtures.
Materials and Equipment
For each lab group (2-3 individuals)-2 lab groups per lab bench:
Electrophoresis chamber
Power supply (shared per lab bench)
Micropipettes and tips to load dye samples.
Various dye mixtures in vials:
Green
Blue
Red
Yellow
Unknown X
Unknown Z
Agarose gel (melted) (front table)
Buffer solution
Hot water bath or microwave to keep the agarose melted (front table)
Pipetting Technique
1.
2.
3.
4.
5.
6.
Depress the operating button to the first stop.
Immerse the tip slightly and release the operating button slowly. The tip should be filled.
Withdraw the tip from the solution, and wipe any drops from the outside of the tip.
Deliver the liquid by gently depressing the operating button to the first stop. Then after a delay
of about a second, continue to depress the operating button all the way down to the second stop.
Withdraw the tip.
Release the operating button to the ready position.
**DO NOT LAY THE MICROPIPETTE FLAT WHEN IT HAS A TIP ON IT! THIS COULD
CONTAMINATE THE PIPETTE
Procedure for Day 1:
Step 1 - Prepare the agarose gel
Check off each step as it is completed during the lab.
o Remove previous classes’ gel by carefully removing the white comb and black casting dams. Be
gentle, because the gel is delicate and can break easily.
o Place gel into baggie.
o Put the black rubber dams on the sides of the gel bed. Be careful to work them on slowly so you
don’t damage the bed. The large portion of the dam goes on top.
o The gel will be melted and can be found in the hot water bath next to the classroom sink.
Careful, it may be very hot!
o Carefully pour heated agarose into gel bed with the black dams on either side until the gel comes
close to the top (about ½-3/4 of the way full). Do not overfill.
o Place the white comb in the center of the gel (it will simply rest on top of the bed). Make sure it
is even & straight.
o Put the gel bed out of the way and wait for the agarose gel to solidify. Do not move it until it has
hardened. The next class will remove & save your gel.
Step 2 – Practice pipetting samples into wells
Check
o
o
o
off each step as it is completed during the lab.
Pick up one sample from the front lab bench (R, B, G, or Y).
Re-read the section titled “Pipetting Technique” on page one of this lab.
Allow each group member to practice pipetting a sample into the practice wells in the Petri dish.
If properly done, all of the sample will be seen in the well.
o You may use the same tip as long as you use the same sample.
o Dispose of used tips in the cup/beaker on the lab bench.
o Return samples and clean up your lab bench. Have cleaned lab bench approved before leaving.
_______ Approval
Procedure for Day 2:
Step 1 – Prepare your gel electrophoresis chamber and gel
o
o
o
o
o
Remove the gel from the bag and return it to the bed (without the dams).
If the electrophoresis unit does not already contain buffer, pour buffer in both wells so that it
covers the center section. If there is buffer in the chamber, make sure the buffer only covers
the center section. If it is too full, dump a small amount into the sink.
Place the gel bed containing the gel into the electrophoresis chamber.
Load your samples, following the directions on page 3.
Check with me that you have the right amount of buffer. _______ Approval
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Step 2 – Load the samples into your gel
o
o
o
o
o
Using a micropipette and new tip for each dye, load 10µL of each sample into the wells in the
middle of the gel.
Do your best to just get the dye in the wells and not in the buffer or on top of the gel.
Add buffer carefully to the side of the chamber so that the fluid level slightly covers the gel.
Attach top of chamber and lock into place.
Do not turn on the power supply until instructed to do so. (Danger- High Voltage)
Step 3 – Run and analyze your gel
o
o
o
o
o
o
o
o
o
o
Connect the electrophoresis unit to the power supply (red to red, black to black). Plug in the
power supply.
Adjust the switches and dials to the correct settings: Select Volts (not milliamps); select Low
(not high).
Let Mrs. Skinner know when you have reached this step and are ready to run your gel.
Before you turn on the power supply, have your setup checked by your teacher.
Turn the unit on (  ). Adjust the voltage dial to 150 V. You will see bubbles being formed, and the
dyes will start moving toward both the negative and positive poles.
Record the time, and electrophorese samples for about 10-15 minutes.
Turn off the power supply, disconnect power cords from the chamber, and remove top of
electrophoresis chamber.
Remove the bed and gel from the chamber, dry the bottom with a paper towel and place gel on
light box.
Write your names on the transparency paper (with the transparency marker) and place it on the
light box with your gel. Photograph your gel with name tag while it is still on the light box using a
cell phone if possible. This should be either emailed to me or if you can-find a way to print it in
color either at home or in the library.
Using colored pencils, draw your gel in the data section of your lab.
Step 4 – Clean up
o
o
o
o
DO NOT DUMP BUFFER!!
Break off unused gel & put into baggie on front lab bench. Dispose of your used gel in the trash.
Wipe down and dry your lab bench.
Have your cleaned lab bench approved before leaving.
_______ Approval
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Names of group members: __________________________________________________________
Data: Use colored pencils to record your results in the diagram below.
RED (+ pole)
Lane
Sample
A
R (red dye)
B
Y (yellow dye)
C
G (green dye)
D
B (blue dye)
E
__(skip)__
F
X (unknown mixture)
G
Z (unknown mixture)
H
________
BLACK (- pole)
Questions
1.
What is the purpose of gel electrophoresis?
2. What are some factors that determine the distance molecules move through the gel?
3. a. Toward which poles (+ or -) on the electrophoresis apparatus did the dyes move?
b. What charge do the dyes have (fyi: this is the same charge as DNA)?
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4. What color dyes (different pigment molecules) make up each of the food colors used in the
experiment? Record the colors that you see in your gel for each of the samples you added.
Red Yellow GreenBlue5. What known dyes were mixed to form the unknown? (Hint: Each unknown is composed of a mixture
of 2 known dyes – red, yellow, blue or green.)
X-
Z-
6. a. What color pigment moved the farthest through the gel? Why?
b. What pigment showed the least movement? Why?
7. What are some practical uses of gel electrophoresis?
8. How does this lab simulate DNA fingerprinting that is done in a crime scene investigation lab?
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9. Below is an example of a DNA fingerprint made from DNA collected at a crime scene and 3 suspects.
Positive (+)
Negative (-)
a. Which suspect can be assumed to have committed the crime?
b. Explain how you came to this conclusion.
c. Where would you find the largest pieces of DNA?
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