Techniques Lab B:

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Class Set
Techniques Lab B:
Electrolysis and Electrophoresis
Background
Before learning about electrolysis and electrophoresis it helps to become familiar with the power
supply and gel box. The electrode at which electrons enter the gel box from the power supply
(along the black wire) is called the cathode and is negative (-). The electrode at which electrons
leave the box and re-enter the power supply (along the red wire) is called the anode and carries
a positive charge (+). The flow of electrons sets up a potential energy difference between the
electrodes. This is known as potential, and is measured in volts. It establishes an electric field
through which the ions in the gel box fluid migrate. The migration of ions in the fluid creates
electrical current which is measured in milliamperes (milliamps).
The splitting of water using electricity is called electrolysis. Electrophoresis is a technique for
separating and analyzing charged molecules (DNA, for example, is negatively charged). The
three main parts of any electrophoresis system are (1) a power supply, which is a source of
negatively charged particles called electrons; (2) a plastic gel box or tank; and (3) a fluid of
water and ions which partially fills the gel box. Recall that ions are atoms that have a positive or
negative charge because they have lost or gained electrons. The migration of ions at different
speeds is the basis of electrophoresis. During electrophoresis, the current splits the water into
hydrogen ions (H+) and hydroxide ions (OH-) (electrolysis).
Purpose
This laboratory explores the principles of electrolysis and electrophoresis, important techniques
used in biochemistry and molecular biology.
Materials per team
power supply
gel box
container for waste (tips)
liquid soap
sodium chloride solution [1M]
300 mL distilled water
Graduated cylinder (10 mL)
phenol red dye solution
P-200 micropipet & tips
P-1000 micropipet & tips
10 mL buffer [1X TAE or TBE]
paper towels
CAUTION

Study the gel box. It is designed so that when the box is opened, the electrodes are
disconnected.

Slide the lid of the gel box off by using the small finger hold "mounds" on the top of the
lid. DO NOT, repeat, DO NOT PULL ON THE WIRE LEADS TO REMOVE THE TOP!!!

Since any wet surface can become conductive, it is advisable NOT to touch any part of
the apparatus (gel box, wires) while the power supply is on. This is especially important if
the outside of the box is wet, or if your hands are wet.
Procedure
As you move through the following steps, observe what is happening inside the gel box!
Record your data on the Activity Sheet.
1. Examine the power supply and gel box and identify all the parts you labeled on your
diagram.
2. With the power supply OFF, connect the empty gel box to the power supply.
3. Turn the power supply ON, and select a potential of 120 volts.
4. Turn the power supply OFF.
5. Open the gel box, and add 300 mL of distilled water. Turn the power supply ON. Observe
the gel box. Turn the power supply OFF.
6. Add 1 mL of the sodium chloride solution to the distilled water in the box. Carefully
"slosh" the box to mix the salt and water. Turn the power supply ON. Observe & record.
When current is flowing, the chemical reactions at the cathode and anode indicate that
differences in pH develop at either end of the box:
At the cathode:
At the anode:
4 electrons- + 4 H20 ---> 2 H2 (gas) + 4 OH-.
4 H20 ---> O2 (gas) + 4 H+ + 2 H20 + 4 electrons-
(Write these equations on the bottom of your lab handout. You’ll need it to answer questions.)
7. To verify that a change in pH is occurring during electrophoresis, we will use an indicator
dye called phenol red. CAREFULLY slosh the water and salt solution in the gel box to
mix them and then add 100 µL of phenol red. Keep sloshing until the liquid in the gel box
is a uniform color. What is the color?
8. Now turn the power supply ON. Record the time it takes for color changes to take place.
a. The accumulation of OH- makes the solution basic. This is confirmed by the
phenol red remaining pink.
b. The accumulation of H+ makes the solution acidic. This is confirmed by the phenol
red turning yellow.
9. Turn the power supply OFF.
10. Add 10 mL of buffer solution. Slosh well to mix. What color is the solution?
11. Turn the power supply ON again. Record the time for color changes, if any. What might
the lack of a color change tell us about the effect of buffer on pH?
Upon completion of this lab




Dispose of designated materials in the appropriate places.
Leave equipment as you found it.
Check that your work station is in order.
Wash your hands.
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