Gel Electrophoresis Lab
Objective
Compare DNA samples from two suspects against DNA taken from a crime scene. Can you identify which
suspect committed the crime using these DNA fingerprints?
Practice loading samples
1. Set the micropipette to 15 µl. To change the volume of the BLUE micropipette, twist the knob under the
plunger until you have reached your desired volume. Push the plunger down once to make sure this new
volume is “set”.
2. To add a tip to the micropipette, push the micropipette down onto a tip. You should not have to use your
hands to accomplish this. Please do not use too much force to put the tip on the micropipette; these instruments
are expensive.
3. Load a 15 µl sample into the practice gel. To do this, press the plunger of the micropipette until you reach
the first stop. Place the tip of the micropipette into the methylene blue solution present in the microtubules, and
release the plunger to draw the liquid into the tip. Using both hands to steady yourself, place the tip of the
micropipette into the MIDDLE of the well and slowly press the plunger until you reach the first stop. To make
sure all of the sample is in the well, press the plunger to its second stop. WHILE KEEPING THE PLUNGER
DEPRESSED, remove the tip from the well.
4. Remove the tip from the micropipette. On the BLUE micropipette, press the ejector down. You should not
have to use your hands to remove tips.
5. Always store your micropipettes in an upright condition.
6. Once everyone has practiced loading the gels, rinse the methylene blue out of the wells in the sample gel.
DNA Forensics
In this experiment, two samples of DNA (one sample from Suspect X and one sample from Suspect Y) are
compared to a sample of DNA removed from a crime scene. All the samples are cut with two different
restriction endonucleases and the DNA fragments are then separated using gel electrophoresis. The banding
patterns created are compared to determine which suspect matches the DNA evidence left at the crime scene.
Procedure A: Load the Gels
1. Load the samples into an agarose gel. Remember, these gels are much more fragile than the practice gels
you used earlier, so be very careful NOT to pierce the wells of your gel while transferring samples. Follow the
instructions below to load your samples into the wells of your agarose gel.
2. Remove the casting tray from the electrophoresis chamber. Do this carefully so that your gel does not slip off
the tray. Place the tray on a paper towel on the lab bench. This will help you see the wells a bit better.
3. For the Forensics Lab, you will load your samples in this order. There are two samples from each subject
since two different endonucleases (enzyme #1 and enzyme #2) were used to cut the DNA.
a. Place sample SX1 (Subject X) in well #1.
b. Place sample SY1 (Subject Y) in well #2
c. Place sample E1 (Evidence) in well #3.
d. Skip well #4.
e. Place sample SX2 (Subject X) in well #5.
f. Place sample SY2 (Subject Y) in well #6.
g. Place sample E2 (Evidence) in well #7.
Unfortunately, you cannot skip a well in between each sample, so just load the samples in this order.
4. Set your micropipette to 15 l. Transfer your samples to the appropriate wells. USE A NEW TIP FOR
EACH SAMPLE! Place your old tips in a beaker. At the end of lab, dispose of these tips in the garbage.
5. Once all of your samples are loaded, GENTLY place the tray back into the chamber. MAKE SURE YOUR
WELLS ARE ALIGNED WITH THE BLACK END (anode or negative end) OF THE CHAMBER!
DNA is negatively charged so it will migrate TOWARD the red end (cathode or positive end) of the
electrophoresis chamber during electrophoresis.
6. Place your gels into the buffer gently so that your samples do not slosh out of the wells. The loading dye
is heavy but if you jostle your gels too much, your samples will get rinsed out of your wells.
Once your samples are in their respective agarose gels, proceed to Procedure B to finish the lab activity.
Procedure B: Electrophorese
1. Before closing the top of your electrophoresis chamber, make sure that the buffer is completely covering
your gel. Close the top of your chamber.
2. Plug the black line into the black hole and plug the red line into the red hole that sits directly below the black
hole. Make sure you plug your lines in correctly! The lines sit vertically once plugged in with the black line
sitting above the red line.
3. Make sure the wells in your gel are near the black end (anode or negative end) of the chamber.
Remember, DNA is negatively charged so it will migrate toward the red end (cathode or positive end) of the
chamber during electrophoresis.
4. Turn your power supply on. Make sure your voltage reads between 125 and 145 volts. Let’s try to run all the
gels below 145 volts as too much voltage may cause the gel to overheat. The knob on the bottom should be
toggled to low. Have your instructor check your set up once you get started. You should see bubbles at the
black end of the chamber once you start the electrophoresis.
5. Eventually, you will see the loading dye migrate toward the opposite (red) end of the chamber. The dye will
resolve into two bands: the faster moving purplish band is bromophenol blue while the slower moving aqua
band is xylene cyanol. Bromophenol blue migrates through the gel at the same rate as a DNA fragment
approximately 300 base pairs long. Xylene cyanol migrates at a rate equivalent to a DNA fragment
approximately 4,000 base pairs long.
6. When the bromophenol blue dye has reached the opposite (red) end of the gel, turn your power supply off
and let the gel sit for a minute or two. Gel electrophoresis takes about 45-75 minutes, depending on the
concentration of agarose in your gel and the amount of stain in the gel. The light stain in the gel should
combine with the DNA while it separates so that you can see faint lines representing different sized DNA
fragments.
7. Carefully remove the casting tray and slide your gel into a staining tray labeled with your names. Take your
gel to your instructor for further staining.
Results and Discussion
1. Look at the gel. Which suspect’s DNA (Suspect X or Suspect Y) matches the evidence DNA?
2. If the two suspects had been identical twins, how would it have influenced the results?