Lab 305: Part IV, Day 2 and 3
The term 'electrophoresis' literally means "to carry with electricity." It is a technique for separating and analyzing mixtures of charged molecules. Due to its sugar-phosphate backbones, DNA is a negatively-charged molecule. When placed in an electric field, it will migrate toward the anode (+). The speed of migration of DNA in an agarose gel depends on the size of the piece; small pieces experience less resistance and move faster (farther) than the larger pieces.
- Remember, it is good practice to turn the power supply OFF before touching or opening a gel box.
- If two teams are connecting their gel boxes to one power supply, be sure to communicate with each other whenever the power supply is turned ON or OFF.
C O N V E N I E N T S T O P P O I N T.
- Apply a drop of liquid soap to the inside of the gel box LID and spread evenly. WITH THE POWER SUPPLY OFF, slide the lid onto the gel box and connect electrical leads anode to anode (red to red) and cathode to cathode (black to black). Both electrodes should be connected to one power supply channel.
- Turn the voltage knob down to ZERO. Then turn the power supply ON. Set the power supply to about 100 Volts.
- Notice there is a switch to direct the LED display to read in either volts or milliamps. Use it to verify how much current is flowing through the gel. (It should read about 40 milliAmps with one gel box hooked up or 80 milliAmps with two boxes hooked up with 1XTAE buffer.)
- Shortly after the voltage is applied, you should notice something happening at each electrode... what is it? Allow electrophoresis to proceed until the dye, and DNA, are out of the wells and safely into the agarose matrix.
- Turn the power supply OFF and disconnect the leads.
- Remove the casting tray from the gel box. CAREFULLY slide your gel off the casting
tray and into an empty plastic tray or "boat." Label the rim of the tray with your initials.
Place the gel and "boat" into a Ziploctm baggie and seal. Your teacher will instruct
you how to store the gels overnight.
- To resume electrophoresis, CAREFULLY slide your gel back onto a casting tray. BE SURE YOUR DNA WILL BE GOING IN THE CORRECT DIRECTION! Be sure the casting tray's gates have been lowered. Set the tray into a gel box containing buffer; connect the gel box to the power supply; turn the power supply ON and adjust the voltage as instructed above.
- Continue to electrophorese until the fastest-moving dye front has advanced at least 2/3 to 3/4 of the way across the gel (about 45 minutes time).
- Then, turn the power supply OFF and disconnect the leads.
- Remove the casting tray from the gel box. CAREFULLY slide your gel off the casting tray and into its labeled plastic tray or "boat." Your teacher will instruct you how to store the gels overnight.