1-3 Electrophoresis
Name: _____________________
Background information:
The purpose of agarose gel electrophoresis is to separate charged biological
molecules. It can also be used to determine the size of the separated molecules. The
separation medium for this procedure is the agarose gel. Agarose is a highly purified
polysaccharide molecule made from agar. Agar comes from processed seaweed and
is non-toxic. The agarose gel is placed in the horizontal gel electrophoresis
apparatus. This is a plastic box with electrodes at either end. The electrodes are
pieces of platinum wire, which are fragile, so care should be taken when handling
the apparatus to avoid damaging these wires.
When the agarose gel is prepared, a “comb” is used to make small pockets
(called wells) in the gel. The samples to be separated will be placed in these wells.
Agarose by itself will not conduct electricity and it tends to dry out when exposed to
air. Once the agarose gel is prepared, it is placed in the apparatus and then covered
with a solution (called the buffer). The buffer solution contains ions, which allow it
to conduct electricity. It also controls the pH. The next step is to “load” the samples.
Samples are loaded by placing small amounts of them into the wells with a
micropipet or a transfer pipet. The samples are mixed with glycerol to increase the
density of the sample material. This prevents the samples from floating out of the
wells.
Once everything is assembled, the apparatus is connected to a power source
and electrical current is applied. One end of the apparatus becomes the anode
(colored red) and the other end is the cathode (black). The anode is the positive
electrode, making the cathode the negative electrode. This experiment uses dyes
instead of DNA as the samples to be separated. Once the current is applied, the dye
molecules begin to move through the wall of the well into the agarose gel. As the dye
molecules have a net negative charge, they move toward the anode. The agarose gel
contains microscopic open spaces, called pores. These pores act as a molecular
sieve. Smaller molecules move through the spaces more easily than larger
molecules, so the smaller molecules move faster through the gel. After a sufficient
length of time, the molecules will separate based on their size and net charge.
In order to determine the size of the separated dye molecules, it is necessary
to run a standard sample along with the dye samples of unknown size. The size of
the standard sample molecules is known, so the size of the unknown molecules can
be determined by comparison. Size determination is achieved by plotting the
distance the molecules move through the gel on a graph. A standard curve is plotted
by measuring the distance the known molecules move and using the size of these
molecules which is given in the handout. The size of the unknown molecules can be
determined by comparing the distance each one moves to the standard curve.
Now watch the video at the following site:
https://www.youtube.com/watch?v=mN5IvS96wNk
Pre-lab Questions:
1. What is the function of gel electrophoresis?
2. Make a diagram illustrating how gel electrophoresis works.
3. a. If the molecules are negatively charged which direction are they going to travel?
What if they are positively charged?
b. Not all molecules are attracted to the charged terminals at the end of the
electrophoresis chamber. What can you speculate about the nature of these
molecules?
4. What is the purpose of the wells in the gel?
5. After a short period of time of running your gel, you will begin to see bands, why
do they begin to separate?
6. If your bands are too close together what should you do to allow they to have
more space between them?
7. What is the purpose of the standard that you will put in the first well of the gel?
8. Explain why glycerol is added to the sample solutions before they are loaded into
the wells.
Procedure
Today you are going to use the agarose gel that your group made in the last class to
separate the candy coating on M&Ms and Skittles into their individual components.
First we need to obtain the dye in the candy coating.
Part I: Obtaining the Dye Samples
Part II: Loading the Gel
5. Using a separate tip for each sample, load 10 micrometers of your
reference dye and candy dye extract into the gel, with one sample per well.
The standard is loaded in the well at the left hand corner of the gel.
Lane 1: Standard
Lane 2: Candy 1 dye extract
Lane 3: Candy 2 dye extract
Lane 4: Candy 3 dye extract
Lane 5: Candy 4 dye extract
Post lab questions
1. Why was it necessary to place the comb on the end of the gel closest to the black
electrode?
2. What two factors control the distance the colored dye solutions migrate?
3. What component of the electrophoresis system causes the molecules to separate by
size? Explain.
4. What force helps move the dyes through the gel?
5. The gel used for this experiment is 0.8% agarose. If you needed to separate
smaller molecules than the ones used here, what change would be necessary in the
gel? Explain your answer.
6. What was the purpose of running the standard dye sample?
7. Agarose electrophoresis is commonly used to separate DNA molecules. Explain
how you expect a mixture of DNA fragments containing lengths of 350 base pairs,
250 base pairs and 75 base pairs to separate. (You can draw a picture if that helps.
And you can refer back to the Khan Academy video if you are stuck!)
8. The apparatus pictured below is used to separate DNA. Each DNA sample is a
mixture of different size pieces of DNA. Four different samples (labeled A, C, G, and
T)
are separated in the picture above. On the next page, explain how this apparatus
works in separating molecules of different sizes and charges.
If you want to do more with electrophoresis, there is a really neat virtual
lab at the following site:
http://learn.genetics.utah.edu/content/labs/gel/