Name:_____________
Dye Gel Electrophoresis
Molecules can be separated by charge and size using a technique called gel electrophoresis.
How does gel electrophoresis work?
An agarose gel is made by dissolving agarose powder in a buffer solution. This mixture is
heated and poured into a mold. A plastic “comb” is inserted to create wells, or depressions in
the gel. The wells are where the molecule samples will be placed.
The gel is placed into the electrophoresis chamber such that positive (red) and negative (black)
electrodes are at each end. A buffer solution is added to cover the entire gel. This buffer has
ions in it to conduct an electrical current. Since the gel is also made with buffer, it also has the
ability to conduct a current. Once the samples are loaded into the wells, the chamber is
connected to a power source, and the voltage is turned up. This power source is what creates
the negative and positive electrodes.
Since opposite charges attract, the negatively charged molecules in the samples will start to
migrate across the gel towards the positive electrode, and positively charged molecules will
migrate towards the negative electrode. If a sample is composed of a mixture of molecules of
different sizes, the different size molecules will migrate at different speeds across the gel.
Larger molecules will be slowed down by the gel and move more slowly than smaller molecules.
Comparing the resulting bands and their distances from the wells, allows you to compare sizes
and charges.
Here is an example dye gel:
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Use the gel to answer the following questions.
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1. Why are the wells placed in the center of the gel rather than at one end?
2. Which dyes are positive dyes? How do you know?
3. Which dyes are negative dyes? How do you know?
4. Which dyes are made of the largest molecules? Smallest?
Are you ready to run your gel???
Procedure:
We will use gel electrophoresis to determine the charge of various dyes. We will also
determine the composition of two dye mixtures.
CAUTION: The materials and equipment used in this lab are extremely expensive. It is
important that you understand what you are doing before running the gel. Any
student found using materials and/or equipment in any way other than as directed by
your instructor may result in failure of participation in this lab.
1. Obtain an agarose gel from your instructor. Carefully support it as you take it to your lab
station as it is fragile and make break.
2. Place the gel holder in the chamber. The red side of the holder should be on the red side
of the chamber. The black side of the holder should be on the black side of the chamber.
This will help you remember which side is positive (red) and negative (black).
3. Place the gel on the gel holder, and obtain approximately 400mL of TBE buffer solution
from a stock solution prepared by your instructor. Use a beaker to measure the buffer
and carry it back to your lab station. Pour the solution into your chamber. The buffer
should cover the entire gel.
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Figure 1. Dye location in wells.
4. Use figure 1 to sketch which dyes will go in which wells. Use the micropipette to
obtain a 10ul volume from one dye sample. The micropipette is preset to 10ul. Load the
dye into the first well as demonstrated by your instructor. Be sure to immerse the
micropipette tip into the buffer solution before loading the dye into the well. This helps to
ensure that there are no air bubbles in the tip.
5. Discard the micropipette tip into your “trash” cup. Put a new tip on the micropipette, and
load the other dye samples into the remaining wells, replacing the tip between samples.
6. Place the cover on the chamber and connect the electrodes to the power pack.
Remember black connects to black and red connects to red. Turn the power on and set
the voltage to maximum. Why do we set the voltage on maximum?
7.
Label a plastic dish with your group members’ names.
8. The gel will run for approximately 15 minutes.
9. Turn off the power, and remove your gel from the chamber.
10. Draw an EXACT copy of your gel on figure 2.
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Figure 2. Banding patterns of dyes.
1. Why did each dye only produce one band, whereas the mixtures produced more than
one band?
2. Which dyes were negative? Positive?
3. Which dye is made of the largest molecules? Smallest?
4. Why are some bands darker than others?
5. What dyes are in Mixture A and Mixture B?
6. If DNA was cut up into different size fragments, how could gel electrophoresis be helpful?