Agarose Gel Electrophoresis: Experiment
Name __________________
Lab Group______________
Edible Colors
Purpose
To introduce the principles and terminology of electrophoresis and demonstrate the
separation of food color dyes with agarose gel electrophoresis.
Materials List
Electrophoresis Chamber
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1 plastic dish
1 slide box lid or bottom
Aquarium sealant
Large needle
Seizing wire
Hot glue gun and glue
Power Supply
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5 nine-volt batteries
1 nine-volt battery clip
2 alligator clips (1 red / 1 black)
Scissors or wire cutters
Agarose Gel
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Transfer pipettes
2” x 3” glass slide
Well comb (template and Styrofoam)
Tris-Borate-EDTA Buffer (TBE)
Agarose
Graduated cylinder
Erlenmeyer flask
Balance
Microwave or hot water bath Sample Preparation
Small vials or tubes
Tube rack
Permanent marker
Capillary tubes with bulb
Food colors
50% glycerol solution
Safety Precautions
Agarose Gel Electrophoresis: Experiment
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Chemicals – Aquarium Sealant, Agarose, and TBE Buffer are all irritants. Do not allow
these chemicals to come into contact with your body. If one does come into contact with
your body, flush the site of exposure with water
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Glassware – if you use glass beakers, graduated cylinders, and/or flasks, you need to
take special caution to not break them. If you do break any glassware, remain calm, alert
your teacher, and follow your lab safety protocol to carefully dispose of it in an
appropriate manner.
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Electricity – do not touch the alligator clips or buffer when the power supply is
assembled and hooked up. IT WILL ELECTROCUTE YOU!!!
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Hot Objects – the hot glue gun and the agarose solution will both be hot. Do not touch
them or you will burn yourself. Use a hot pad or tongs to move the flask of agarose until
it cools. If someone gets burned, rinse the burn with cool water and then follow your
classroom or laboratory protocols for seeking medical attention if necessary.
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Sharps – the needle, seizing wire ends, and capillaries are all sharp. Be careful when
handling these items and dispose of them properly.
Building the Electrophoresis Chamber
1. Place the slide box in the center of the plastic dish and trace around the edge with the
permanent marker.
2. Extend the lines of the long sides of the slide dish all the way up one side of the plastic
dish. Use the permanent marker to place a spot near the rim of the plastic dish, about 1
cm out from each of the lines.
3. Carefully use the large needle to punch a hole through the plastic dish at each spot.
4. Use the scissors or wire cutters to cut two pieces of seizing wire about 10 cm longer than
the length of the plastic dish.
5. Take one piece of wire and make a 90˚ bend 1 cm from the end. Be careful because the
ends of the wire will be sharp.
6. Go down the wire and make another 90˚ bend so that the wire will fit down into the
plastic dish and touch both sides.
7. Position the wire 1 cm above the bottom of the plastic dish (the width of your finger) and
make a 90˚ bend that will allow the wire to come out of the hole.
8. Feed the end of the wire through the hole and bend it into a loop. Cut off and dispose of
any excess wire.
9. Repeat these steps with the second piece of wire and the second hole.
Agarose Gel Electrophoresis: Experiment
10. Place the slide box in the center of the plastic dish to make sure that neither of the wires
touches it. Make necessary adjustment to prevent the wires from touching the slide box.
Remove the slide box.
11. Plug in the hot glue gun and allow it to heat up. Be careful when using the hot glue gun
because the gun and the glue will both get very hot.
12. Use the hot glue to secure the wire at each of the 90˚ bends. Allow the glue to dry.
13. Put a thick bead of aquarium sealant around the rim of the slide box. Place the slide box
open side down into the center of the plastic dish where you traced it. If necessary add
more sealant around the edges to make sure there will be no leaks. The aquarium sealant
will need to dry overnight.
Building the Power Supply
14. Cut the battery clip in half with the scissors, be careful to not cut the wires.
15. Using the scissors, strip away about 1.5 cm of plastic covering off the wire attached to
each half of the battery clip.
16. Pull the plastic cover off one arm of each of the alligator clips. Slide the plastic cover
onto the wire.
17. Wrap the wire from the negative end of the battery clip (red wire) around the arm of red
alligator clip.
18. Slide the plastic cover back onto the alligator clip to cover the connection.
19. Repeat these steps for the black wire and black alligator clips.
20. Build a pyramid with the batteries, three on bottom and two on top. Connect the negative
ends of the batteries to the positive ends of the opposing batteries.
21. When you are ready to use the power source attach the red wire to the empty positive end
of the battery and the black wire to the empty negative end of the battery.
22. Do not touch the alligator clips on the end of the power supply or the buffer when the
power supply is attached to the electrophoresis chamber because it will electrocute you.
Store power supply with the batteries taken apart.
Preparing the Gel and the Samples
Agarose Gel Electrophoresis: Experiment
23. For this experiment a 1% Agarose gel was used. Only a few milliliters of agarose gel
solution are needed per gel. Here is the recipe for 10 ml:
a. Weigh 0.1 g of agarose powder on the balance
b. Place it in the Erlenmeyer flask
c. Add 10 ml of 1X TBE
24. The agarose and TBE will need to be heated in order for the agarose to dissolve. It takes
about 1 minute in the microwave, or you can place the flask in a hot water bath. When
the agarose is melted, the solution will be clear and there will be no particles floating in
it. Caution: the flask and the liquid will be very hot! Handle with care.
25. Allow the agarose solution to cool. It will be ready when the bottom of the flask is cool
enough to touch without burning your skin.
26. While the agarose solution cools, take the glass slide and lay it on a hard, level surface.
27. Take the two binder clips and the well comb. Grip the well comb in the two binder clips
so that the comb is level and just above the glass slide.
28. Take a transfer pipette and fill it with the agarose solution. Starting at the comb and
moving back, lay the agarose solution on the slide. Be sure to: work quickly; do not
allow any gaps; do not over fill the slide because the agarose will spill over the edge; and
pop any air bubbles immediately.
29. Allow the gel to cool and solidify. The gel will turn opaque when it is set. (About 5
minutes)
30. While the gel is cooling, prepare the samples.
31. Label the test tubes/vials with the color of food dye that will be put in them. Label one
for red, blue, green, and yellow.
32. In each test tube place one drop of food dye and three drops of 50% glycerol. Repeat this
for each of the samples.
33. Remove the comb from the gel by gently pulling straight up.
34. When moving the gel, be very careful to hold the slide flat. If you tilt the slide, the gel
can slip off.
Loading and Developing the Gel
35. Place the gel on top of the slide box in the electrophoresis chamber. Make sure that the
slide is positioned so that the row of wells is parallel with the wires.
Agarose Gel Electrophoresis: Experiment
36. Pour enough TBE buffer into the electrophoresis chamber so that it covers the gel. Do
not pour the TBE directly onto the gel; pour it to the side of the gel in the plastic dish. If
the TBE is poured directly on the gel, it could push the gel off of the slide.
37. Decide what sample you are going to put in each well and write it down on your
worksheet.
38. Gently put the bulb on a capillary tube. Be very careful when handling the capillary
tubes because they are very thin glass tubes and they break easily. To fill the capillary
tube, place the tip of it in the sample and it will automatically draw the sample up. Be
careful to keep the sample in the capillary tube and do not let it into the bulb. Once the
tube is almost full you will need to cover the hole on top of the bulb and squeeze the bulb
just a little to keep the sample from coming up into the bulb. Hold the pressure steady so
that you do not push the sample out of the tube. This may take a little practice.
39. Place the tip of the capillary tube into the buffer right above the well you are going to fill.
Do not put the capillary tube into the well because, if it is bumped, it could punch
through the well leaving a hole that would leak out the sample. Slowly squeeze the bulb
more to push the sample out of the capillary tube. The sample is heavier than water
(because you added the glycerol), so it will go straight down into the well. Some of the
sample may come out of the well and get into the buffer. That is all right as long as most
of it is in the well. Dispose of the capillary tube in a container designated for glass
disposal.
40. Repeat this step with a new capillary tube for each of your samples.
41. After all of the samples are loaded, assemble your battery pyramid and connect the wires
to the batteries.
42. Clip the black alligator clip to the loop on the wire that is behind the wells and the red
alligator clip to the loop on the wire that is in front of wells. Do not touch the buffer
while the clips are attached! You will be electrocuted!
43. Watch for bubbles to form on the wires in the TBE. This will tell you that the current is
flowing.
44. Allow the gel to develop until you can see the colors separate. The food dyes in the food
colors will travel through the gel with the electrical current towards the red wire.
45. When the gel is done developing, unclip the alligator clips and take the battery pyramid
apart.
46. Remove your slide from the electrophoresis chamber and place it onto a white paper
towel or piece of paper so you can see the colors better.
47. Pour the TBE buffer into the sink.
48. Observe the results of the gels.
Agarose Gel Electrophoresis: Experiment
Name __________________
Lab Group______________
Edible Colors
Before Experiment
1. What is electrophoresis?
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2. What is an example of a use for electrophoresis?
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3. What is a mixture?
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4. What is a charged molecule?
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5. How does electrophoresis separate a mixture of charged molecules?
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6. What is agarose derived from and what is it commonly used for?
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7. What is TBE used for in this experiment?
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8. Are there any safety precautions you need to take when working with the
agarose and TBE?
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9. Are there any safety precautions you need to take when developing the gel?
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10. What dyes do you think each of the food colors are made of?
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11. What do you think it will look like when all of the colors separate?
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12. What sample did you put in each well?
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Agarose Gel Electrophoresis: Experiment
During Experiment
13. What do you see after 5 minutes?
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14. What do you see after 10 minutes?
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15. What do you see after 20 minutes?
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16. What do you see after 30 minutes?
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After Experiment
17. What dyes do each of the food colors separate into?
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18. Did any of the colors contain dyes that you did not expect?
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19. What dye moved the farthest? Why did this dye move farther than the rest?
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20. What dye moved the least farthest? Why did this dye not move as far as the rest?
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Agarose Gel Electrophoresis: Experiment
21. Is the yellow dye in the yellow food color and green food color the same?
How do you know?
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22. Are there any other food colors that have the same dyes?
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23. What two things help determine how far each dye moves?
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24. If all of the dyes are attracted to the positive electrode, what is the overall
charge of the dyes? Positive or Negative? Why?
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