DNA Fingerprinting

Overview

In this activity, you will use gel electrophoresis to analyze six DNA samples. You will prepare your own agarose gel, load the DNA 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
  • 6 DNA samples labeled A, B, C, D, E, and F
  • micropipette and tips
  • Sybr Safe

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 flask in microwave or on heat until agarose is melted.
    • Stop periodically and swirl solution and do not permit to boil over.
  3. Assemble the casting tray by blocking the ends with the gaskets.
  4. Place the comb into the casting tray at the NEGATIVE end.
  5. The instructor will add 6μl Sybr Safe to your gel solution at this time.
  6. Place the casting tray inside the refrigerator and pour the solution into the tray.
  7. Wait until the gel is solidified.
  8. Carefully separate the gaskets from the tray.
  9. 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.
  10. Completely cover the gel with TBE buffer.
  11. 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.
  12. Cover the electrophoresis chamber with the lid and ensure good contact between electrodes.
  13. 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.
  14. After 40 minutes, stop the current and remove the gel in casting tray.
  15. Your instructor will slide the gel onto a UV transilluminator behind a shield and show the results to the class
    • Document the findings of the gel by photographing with your phone.

Analysis

Work with your lab group to answer the following:

  1. Why are the samples loaded at the negative side of the gel?
  2. What is the role of the dye in these samples? Should we be alarmed that the samples are all the same color?
  3. What does it mean when there are multiple bands in a lane? What does it mean when there is only one band in a lane?
  4. What can we conclude from the banding patterns in this forensics or paternity case? Is this sufficient data for these conclusions?
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