DNA fingerprinting (RFLP)

DNA Fingerprinting (activity)

  1. The origin of the DNA samples for this exercise will be explained by the Instructor as numerous scenarios may be used
  2. Prepare a 1% agarose gel by adding 60ml Tris-Borate-EDTA buffer (TBE) to 0.6g agarose in an erlenmeyer flask
  3. Place flask in microwave or on heat until agarose is melted
    • stop periodically and swirl solution and do not permit to boil over
  4. Assemble the casting tray by blocking the ends with tape or plastic gaskets
  5. Place the comb into the casting tray at the NEGATIVE end
  6. The instructor will add 6μl Sybr Safe to his/her own gel solution at this time
  7. You may place the casting trays inside a refrigerator and pour the solution into the tray
  8. Wait until the gel is solidified
  9. Carefully separate the gaskets from the tray ensuring not to tear apart the wells made by the comb
  10. Remove the comb and place the casting tray into an electrophoresis chamber
  11. Cover the gel with TBE buffer
  12. Using a micropipettor, load 40-50μl dye samples sequentially into  the wells
  13. Cover the electrophoresis chamber with the lid and ensure good contact between electrodes
    • It is conventional that the POSITIVE side of the tank is nearest to you
    • With the POSITIVE side nearest to you, load the samples from left to right
  14. 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
  15. After 40 minutes, stop the current and remove the gel in casting tray
  16. Slide the gels into the staining solution if they do not include Sybr Safe for visualization the subsequent meeting time
  17. The 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
    • The instructor will discuss the results and ask for you to interpret the findings

Activity Follow-up

  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 that there are multiple bands in a lane? What does it mean that 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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