Program: Wildlife Forensics
Gel Electrophoresis – Protocol
Preparing the Gel
1. Set up the tray by firmly attaching the rubber stoppers
to either end. Place the well template at one end.
Set aside.
2. Use the heat gloves to obtain a small flask of agarose
gel in liquid form from the water bath. CAUTION –
Use gloves, the glass is hot.
3. Carefully pour the gel into the prepared tray; the gel
should not be above the top of the rubber
stoppers. Let cool for about 20 minutes or until
the gel completely solidifies. Set the gel aside to
practice pipetting.
Practice Pipetting
4.
Gather your materials: micropipette, clean tips, garbage container, cup
of water, gel with practice wells, gel
electrophoresis kit
5.
Hold the pipette correctly in your hand against
your palm with your thumb on the trigger.
6.
Set the correct volume by setting the knob to 35
μl. Never go less than 10 μl or greater than 90
μl.
7.
Select a new tip for the micropipette, each tip is
only used once. Firmly attach the tip without
touching the tips with your fingers.
8.
Press the top button with your thumb until you feel
the first bit stop. Keep your thumb in that
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Program: Wildlife Forensics
position and place the tip in the liquid. Release the button and the
micropipette will pull the liquid into the tip.
9.
Carefully place the tip of the micropipette in the designated well and
press down to the second stop. Do not release the button until the
micropipette is out of the well.
10. Press the ejector button to discard the tip into the garbage container.
11. Repeat 3-4 times until your feel confident with the movements. Make
sure each person in the group is confident.
12. Throw the gel into the garbage container.
Running Gel Electrophoresis
13. Remove the rubber stoppers from the
prepared and hardened gel.
14. Gather your materials: DNA quick strips,
buffer solution
15. There are six samples to test today: 2 from
a wild chinchilla, 2 from a domesticated
chinchilla, and 2 from suspected chinchilla fur. There are two
different samples from each individual so that two restriction
enzymes could be used. Set up as shown:
a. Wild chinchilla 1
b. Wild chinchilla 2
c. Domesticated chinchilla 1
d. Domesticated chinchilla 2
e. Unknown fur sample 1
f.
Unknown fur sample 2
16. Load your gels at the negative end because
DNA is negative and will move toward the positive
end.
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Program: Wildlife Forensics
17. Use your new pipette skills to carefully transfer each DNA sample into
the appropriate well. Take care to gently add the DNA to each well
and remember to use a NEW tip for each transfer
18. Cover the gels with buffer solution and place the lid on top. Make sure
both groups at the table are ready and connect gel electrophoresis
machine to power source and turn on.
19. Run the electrophoresis for 20 minutes at a voltage of 125 V.
20. Clean up your station while the gel is running.
21. Give the students the time to set up and run their gel, students will
have the protocol sheet with all of this information.
22. After 20 minutes disconnect the electricity and remove the lid.
Students should carefully remove the gel and sketch what they see,
making sure to label each lane.
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Program: Wildlife Forensics
Instructor Notes
Making the agarose gel – The instructor should do this set up ahead of time,
preferably the day before. Make 3 batches for a class of 30
1. Set up the tray by firmly attaching the rubber stoppers to either end. Place the well
template at one end. Set aside.
2. Measure 3 grams of agarose powder and add that to a 500-mL flask.
3. Use a 10-mL graduated cylinder to measure 7.5 mL of concentrated buffer solution
and add to the agarose powder.
4. Use a 100-mL graduated cylinder to measure 367.5 mL of distilled water and add to
the other materials. The total volume should be approximately 375 mL.
5. Place a magnetic stirring rod in the flask and place on a heating plate. Keep the
mixture on medium-high heat and boil until the solution should be clear and the
agarose completely dissolved.
6. Using heat gloves remove the agarose from the heating plate and let cool to 55 ˚C.
7. Set up 8 practice gels by placing 2 sets of combs in each gel. Pour just enough gel
in each well to cover the well template without becoming higher than the rubber
stoppers.
8. Let the gels set for at least 20 minutes to harden and then store tightly wrapped in
plastic in the fridge until needed.
9. The remaining gel can remain in the flask. Once added to a water bath it will melt
again for usage. Each group will need 30 mL of liquid gel to prepare a gel for
electrophoresis. It is probably a good idea to have a few prepared gels with one set
of wells in case a student created gel does not work out.
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