Electrophoresis of Dyes

Overview

In this activity, you will use gel electrophoresis to analyze five dye samples. You will prepare your own agarose gel, load the dye samples, and run the electrophoresis.

Materials

Each lab group will need the following materials:

  • power source
  • electrophoresis chamber
  • casting tray with comb and gaskets
  • TBE buffer
  • 0.6 g agarose
  • 5 dye samples labeled A, B, C, D, and E
  • micropipette and tips

Method

Note: Your instructor may microwave the buffer and agarose for you.

  1. Prepare a 1% agarose gel by adding 60ml tris-borate-EDTA buffer (TBE) to 0.6g agarose in an Erlenmeyer flask.
  2. Place the flask in microwave and heat until agarose is melted.
    • Stop periodically and swirl solution.
    • Do not permit to boil over.
  3. Assemble the casting tray by blocking the ends with the gaskets.
  4. Place the comb into the center of the casting tray.
  5. Place the casting tray inside the refrigerator and pour the solution into the tray.
  6. Wait until the gel is solidified.
  7. Carefully separate the gaskets from the tray.
  8. Remove the comb and place the casting tray with gel into an electrophoresis chamber.
    • Make sure to line up the charges etched on the plastic tray with the correct side of the tank.
    • It is conventional that the POSITIVE side of the tank is nearest to you.
  9. Completely cover the gel with TBE buffer.
  10. Using a micropipette, load 40-50μl dye samples sequentially into  the wells.
    • With the POSITIVE side nearest to you, load the samples from left to right.
  11. Cover the electrophoresis chamber with the lid and ensure good contact between electrodes.
  12. Set the power supply to 100-120V and press the Run button (you should see bubbles at each electrode) and allow to run for at least 40 minutes.
  13. After 40 minutes, stop the current and remove the gel in casting tray.
  14. Place tray on a white background and document your gel.
    • Take pictures with your phone.

Analysis

Work with your lab group to answer the following:

  1. What colors were the dyes originally before loading into the wells?
  2. How many separate bands of dye are in each well following the run?
  3. What does it mean if there are multiple bands in a lane? What does it mean if there is only one band in a lane?
  4. What does the length of migration illustrate to us about the properties of the dye molecules?
  5. In which direction did the dye molecules migrate? What does the direction of migration indicate about the analytes?
  6. Are there lanes where there are multiple bands of the SAME color?
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