Electrophoresis Lab
Electrophoresis is used in biotechnology as a separation method. Molecules move by
attraction to an electric charge, and typically they move through a porous gel that allows
smaller molecules to move faster than larger molecules. Thus, the typical gel
electrophoresis experiment separates molecules by charge and molecular weight (size)
and shape. Applications of electrophoresis include sorting mixtures of proteins or DNA
molecules for identification purposes (like in forensics). This series of experiments is
designed to deconstruct the process of gel electrophoresis.
For a good animation and illustration of this process, visit
http://learn.genetics.utah.edu/units/biotech/gel/
I.
What Goes on During Electrophoresis?
Let’s explore what goes on in electrophoresis with just the gel box and power supply,
leaving out the gel for now:
When the power is turned on, there is a negative pole (black, cathode) and positive pole
(red, anode). Bromothymol blue is a pH indicator which is yellow in acid conditions,
blue in basic conditions. Predict what will happen to the color of the bromothymol blue
solution at the negative pole and at the positive pole when hydrogen ions from water
cluster at one pole and hydroxide ions from water cluster at the other pole.
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Let’s do some experimenting:
Trial 1
1. Take an empty electrophoresis chamber (box) and fill the two wells in the chamber
with bromothymol blue solution (enough to cover the platinum wire at the bottom of each
well of the unit, but not enough to cover the platform of the chamber between wells).
Place the box on a piece of white paper.
2. Note the color of the indicator dye in the chamber at the start – record in table below.
3. Close the chamber with the top piece, and connect red and black power cords to power
supply (red to red, black to black, same channel).
4. Turn on power, set voltage to about 100 Volts. Leave undisturbed for 2-3 minutes.
5. After a few minutes, note the color changes occurring in the electrophoresis chamber.
Record in table below.
Trial 2
6. Disconnect from power source, open box, and add enough bromothymol solution
to completely cover the platform that connects the two wells. Close box,
reconnect to power supply. Run for 5 minutes. Record results in table. Also
note the relative amounts of bubbles at positive versus negative end – do you see
a difference?
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Trial 3
7. Disconnect from power source, open box, and sprinkle some table salt (just a
pinch) into the fluid, swirl to mix, close box, and reconnect power supply.
Record results in table. Note again relative amounts of bubbles at positive
versus negative end – any difference?
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Trial 1
Starting dye color
Dye color at
negative electrode
(black)
Dye color at
positive electrode
(red)
Trial 2
Trial 3
Analysis of results:
1. Why in the first trial was the color change minimal?
2. Why was the color change more apparent in trial 2?
3. How do you think the addition of salt accounts for the results in trial 3?
4. Was there a difference in the relative amounts of bubbles at each pole? Yes No
If you noticed a difference, which pole had more bubbles? If water is undergoing
electrolysis, it breaks down into hydrogen gas and oxygen gas, hence the bubbles.
How does the formula of water, H2O or HOH, tell you where to expect more bubbles?
5. When you open the box, can you smell the presence of another gas? Any guesses
as to its identity?
II.
Loading Microcentrifuge Tubes (Follow teacher directions.)
III. Using Pipettes (Follow teacher directions.)
IV. Making Gels (Follow teacher directions.)
Equipment and supplies:
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An electrophoresis chamber and power supply
Gel casting trays, which are available in a variety of sizes and composed of UVtransparent plastic. The open ends of the trays are closed with tape while the gel is
being cast, then removed prior to electrophoresis.
Sample combs, around which molten agarose is poured to form sample wells in
the gel.
Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA
(TBE).
Loading buffer, which contains something dense (e.g. glycerol) to allow the
sample to "fall" into the sample wells, and one or two tracking dyes, which
migrate in the gel and allow visual monitoring or how far the electrophoresis has
proceeded.
Ethidium bromide, a fluorescent dye used for staining nucleic acids. NOTE:
Ethidium bromide is a known mutagen and should be handled as a hazardous
chemical - wear gloves while handling.
Transilluminator (an ultraviolet lightbox), which is used to visualize ethidium
bromide-stained DNA in gels. NOTE: always wear protective eyewear when
observing DNA on a transilluminator to prevent damage to the eyes from UV
light.
Go to http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/agardna.html
V.
Agarose Gel Electrophoresis with Dyes
Agarose gel electrophoresis makes use of the electrophoresis chamber and power supply
to separate molecules by charge, with the added feature of a gel. By forcing the
molecules through a gel made of a sieving compound like agarose, you can now
simultaneously separate the molecules by size and shape. All negatively charged
molecules will move towards positive pole, and vice versa, but the smaller molecules will
move through the agarose mesh network much faster than larger molecules. In this
experiment you will use gel electrophoresis to separate different dye molecules. Using
dye molecules allows visualization of molecule movement as the process occurs.
Materials and Equipment
Various dye samples in microcentrifuge tubes:
 Micropipettes and tips to load dye samples
 Electrophoresis units and power supplies
 0.8% agarose gels prepared with TBE buffer - with sample wells in center of gel
Loading the samples
1. Follow data sheet below as to where you will load the specific dye samples (A-F).
Place the gel on its casting tray into the gel box and add TBE buffer to the gel
chamber till there is about 2-3 mm buffer over the top of the gel.
2. Load the numbered dye samples into the wells located in the middle of the
agarose gel. Use a different tip to load each sample dye. Load 10-15 l per well.
3. Connect electrophoresis unit to power supply (red to red, black to black), being
careful not to bump your gel. Plug the power source into an outlet.
4. Turn on the power supply, and set voltage to ~100-125 V. The dyes will start
moving toward either the negative or positive pole according to their charge.
Record your observations:___________________________________________
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5. Electrophorese samples for ~10 minutes. Turn off power supply, disconnect
power cords from the chamber, and remove top of electrophoresis chamber.
Analyzing the gel
1. Before you remove gel from chamber, mark the positive end of the gel by cutting
off a small corner of the gel. Now carefully remove casting tray with gel. Place
gel in large weigh tray for viewing.
2. Mark the gel diagram below with + for positive end and - for negative end.
3. Use the gel diagram below to indicate banding pattern results of the samples - use
colored pencils if available.
WELL
A
B
C
D
E
F
DYE
Orange G
Bromophenol Blue
Xylene Cyanol
Crystal Violet
Methyl Green
Mixture
CHARGE observed
Questions
1. Which dyes have a positive charge? Justify your answer.
2. Which dyes have a negative charge? Justify your answer.
3. Which dyes do you think are present in the mixture sample? How do you know?
4.
Can you hypothesize which dyes are larger or smaller in molecular weight in
comparison to each other?
5. Based on results from your first experiment with electrophoresis, what do you
think might happen if the agarose gel was prepared with water rather than TBE?
VI. A Review of the Chemistry Connections
Electrolysis is a process in which electrical energy is used to bring about
chemical change; the electrolysis of water produces hydrogen and oxygen.
Water is reduced to hydrogen and oxidized to oxygen.
A buffer is a solution in which the pH remains relatively constant when
small amounts of acid or base are added; a buffer can be either a solution of a
weak acid and the salt of a weak acid or a solution of a weak base and the salt of
a weak base.
OIL RIG
Oxidation is the loss of electrons.
Reduction is the gain of electrons.
AN OX
Oxidation occurs at the anode.
RED CAT Reduction occurs at the cathode.
Questions
1. When water is reduced to hydrogen, are electrons gained or lost?
2. Is water reduced to hydrogen at the anode or the cathode? Justify your answer.
3. Which process, oxidation or reduction, always occurs at the cathode of an electrolytic
cell?
Credits: Some of the ideas for this laboratory exercise came from the University of
Arizona Biotech project, CEPRAP at the University of California Davis , Access
Excellence and Colorado State University.