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, negative (-). The electrons 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 know 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 that 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 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 hydroxyl 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 wastes (tips) Liquid soap Paper towels/Kim wipes Sodium chloride solution 300 ml-distilled water Measuring device (10 ml) Phenol red dye solution-200 micro pipette and tips-2000 micro pipette and tips 10 ml buffer [1 x TAE or TBE] You can substitute other measuring instruments (droppers, Pasteur pipettes, regular pipettes-volumes are approximate) 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 holds "mounds" on the top of the lid. 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 is on. This is especially important if the outside of the gel box is wet, or if your hands are wet. PRODECURE As you move through the following steps, observe what is happening inside the gel box! RECORD YOUR DATA ON THE ACTIVITY SHEET THAT FOLLOWS. 1. Examine the power supply and identify On / off switch Plugs for leads (2 sets) Voltage select dial Switch between volts / milliamps Digital display Lo / high switch (not present on all models) Special hint: Apply a small drop of liquid soap to the inside surface of the gel box LID and spread evenly with a paper towel.
This should prevent the lid from fogging up as heat is generated. 2. With the power supply OFF, connect the empty gel box to the power supply. 3. Set the lo / high switch (if present on your power supply) to LO, turn the power supply ON, and select a potential of about 100 V (volts).
Record (on your activity sheet) the current (measured in milli amps) generated in the empty gel box. Does current flow through the air? 4. Turn the power supply OFF. 5. Open the gel box, and add about 300 ml of distilled water. Turn the power supply ON.
With a potential of 100 V, record the level of current in the box. Does current flow through distilled water? 6. Turn the power supply OFF 7. Add 1 ml (or 3-4 droppers full) of the sodium chloride to the distilled water box.
Carefully "slosh" the box to mix the salt and water. Turn the power supply ON. Set the voltage to 100 V. Record the current.
Does current flow through distilled water that contains ions (Na+Cl-)? 8. Choose 3 additional voltages and record the current at each voltage. Turn the power supply OFF. When the current is flowing, the chemical reactions at the cathode and the anode indicate that differences is pH develop at either end of the box: At the cathode: 4 Electrons + 4 H 2 O 'a 2 H 2 (gas) + 4 That the anode: 4 H 2 O 'a O 2 (gas) + 2 H 2 O + 4 Electrons + 4 H+ (H positive) 9.
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 ul of phenol red. Keep sloshing until the liquid in the gel box is uniform color. What is the color? 10. Now turn the power supply ON.
At 100 V, record the current. Record the time it takes for the color 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. 11.
Turn the power supply OFF. 12. Add 10 ml of buffer solution. Slosh well to mix. What color is the solution? 13. Turn the power supply ON again and select about 100 V.
Record the current. Record the time for color change, 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 workstation is in order Wash your hands.