Agarose Gel Electrophoresis with Dyes - Student Handout

Agarose gel electrophoresis can resolve molecules based on charge, size, and shape. In this laboratory you will use gel electrophoresis to separate molecules present in different dyes, and you will see some of the applications of electrophoresis.

Materials and Equipment

• Various dyes in microcentrifuge tubes: 1, 2, 3, 4, 5, and an unknown X.
• Pipettes and tips to load dye samples into gel.
• Electrophoresis units and power supplies
• 0.8% agarose in 1X TAE (melted)
• 1X   TAE for electrophoresis units
• 1 clear plastic ruler
  
  

Protocol

1. Get your electrophoresis apparatus.   Make sure the comb is in place (there should be 1 comb in the middle) and that there are stoppers (black casting dams) at both ends of the gel tray.

2. Pour hot agarose into the gel space until it reaches the top of the gel casting tray.   Let the agarose harden, this takes 5-10 minutes.   Don't touch/move your gel until it's hard.   Why not?

3. When the agarose gel is hard, take out the stoppers and pour TAE solution over your gel so that the gel casting tray is completely covered.   What do you think the TAE solution is for?

4. Gently remove the comb and use a pipet to load each dye into a separate well in the gel. Use a separate pipet tip for each sample.   Draw a diagram on the next page as you load the dyes to help you keep track of which sample went into each well!

Diagram of gel:

5. Run that gel!!   Plug the electrodes into your gel box ( red to red, black to black ), being careful not to bump your gel too much.   Plug the power source into an outlet.   How can you tell your gel is running?   What kind of chemical reaction is this?

6. Electrophorese samples for ~10 minutes. Turn off power supply, disconnect power cords from the chamber, and remove top of electrophoresis chamber.

Analyzing your data

1. Carefully remove casting tray with gel and draw a picture of your gel to scale by measuring the distance traveled by the bands in the dyes.   Measurements are typically done from the leading edge of the well to the leading edge of the dye band. If available, use colored pencils to represent the bands

2. Record which of the bands traveled toward the positive and which toward the negative pole. What does this information tell you about the charges of the dyes?   Of the dyes that migrated towards the positive, which was the largest molecule and which was the smallest?   Of the dyes that migrated towards the negative, which was the largest molecule and which was the smallest?

3. Compare known dyes with the unknowns. Which dyes are present in the unknown?

BIOTECH Project Department of Molecular and Cellular Biology The University of Arizona August 3, 1998 Designed by: Ken Kubo, Ph.D. Last Modified March 1, 2002 Nadja Anderson, Ph.D. nadja@email.arizona.edu http://biotech.biology.arizona.edu