Mystery of the Stolen Cat Food: Teacher Guide

This teacher guide is provided to give sample answers to questions. Most of the questions are open-ended, so students may have correct answers that aren't included in this guide. Finally, although the experiment is set up to yield one correct answer, there are variations in data between students. As long as students examine their data carefully and can justify their answers based on their data, that's science! Data are always right and there isn't necessarily a 'right answer'.

The Scenario:

Fluffy is my white haired cat. She sometimes likes to have her meals on the back porch, so I put her dish of cat food outside. Recently she's been whining and complaining because she's hungry. After doing a little detective work, I realized that another white haired cat has been eating Fluffy's food.

However, there are 2 other white haired cats in my neighborhood and I can't tell which cat is eating Fluffy's food. You are going to help me figure out which cat is the criminal!

Some questions to get you thinking about today's lab:

1. What is DNA and what does it do?

   deoxyribonucleic acid makes you who you are, codes for proteins, etc.

2. What are some characteristics or properties of DNA? double-helix, double stranded, negatively charged, colorless, unique sequence in each individual

3. How can we take advantage of these properties to help us figure out which cat is stealing the food? We can take DNA from different individuals and cut it with restriction enzymes. Because each individual has a unique DNA sequence, the restriction enzymes will cut the DNA into different sizes for different individuals. We can take advantage of the negative charge of DNA to separate out the different sized pieces to determine whose pieces (which cat) matches the saliva sample from the bowl.

4. What tricks can we use to see DNA?

   DNA stain such as methylene blue

Materials

• DNA from cat suspect (#1), cat suspect (#2), Fluffy (#3), and Fluffy's bowl (#4)
• agarose
• Tris-acetate/EDTA solution (TAE)
• micropipette/tips
• electrophoresis apparatus

Procedure:

1. Get your electrophoresis apparatus. Make sure that the comb is placed closest to the black electrode and that there are stoppers at both ends of the gel space.

2. Pour hot agarose into the gel space until it reaches the top of the gel box. Let the agarose harden, which should take about 10 minutes. Don't touch/move your gel until it's hard. Why not? If the agarose moves while it's hardening, it will harden unevenly, making it more difficult for the DNA to move through evenly.

3. When the agarose gel is hard, take out the stoppers and gently remove the comb. Draw a picture of your gel and label which samples are where before you add DNA to the gel. The samples are indistinguishable once they are loaded, so everyone needs to have a drawing of what they will load where before they load their DNA samples. This drawing will be useful during the analysis, once the gels are stained.
   
   
4. Load your DNA SAMPLES into the wells near the BLACK ELECTRODE. Why near the black electrode? Be sure to keep track of which samples you loaded in which lanes. DNA is negatively charged, so to move the DNA into the gel with electricity, the DNA needs to be loaded on the negative or black side - it will then move towards the red. If it's loaded near the red electrode, it will migrate off the gel into the TAE.
   
   
5. Pour the TAE solution into the side farthest from you samples, so that you gels is completely covered plus a little more. What do you think the TAE solution is for? TAE is like saltwater - it conducts electricity, plus it is a buffer to control for pH changes
   

6. Run that gel!! Plug the electrodes into your gel box (red to red, black to black), being careful not to bump your gel too much. Plug the power source into an outlet. How can you tell your gel is running? It bubbles at the electrodes. This is a redox reaction, forming H2 gas at the black electrode and O2 gas at the positive electrode.

Analyzing Your DNA Data

Plug in your gel electrophoresis tray, and after 3 minutes draw another picture.

The dyes in the samples will move to the right (towards the positive red electrode) and separate into light blue and purple. They move to the positive electrode because they are negatively charged. They separate because they are different sizes, purple moves faster and farther because it is smaller (lower molecular weight).

The DNA cannot be observed until the gel is stained because DNA is colorless. We add blue coloring to the DNA samples so you can see the samples as you are loading them.

After 15 minutes draw another picture.

The dyes will separate farther and continue to travel to the positive electrode. Run the gels until the purple travels 2/3 of the way across the gel (about 25-30 minutes).

Once the purple dye has migrated approximately 2/3 of the gel, turn off the power and carefully remove the gel. The gel is very fragile, take care to not break it. You can remove the tray that you poured agarose on to and gently slide the gel into the staining tray. At this point you cannot see the DNA, what can you see and how do the four different lanes compare?

The four lanes should all be the same. You should see two dyes in each lane, purple that migrated the furthest and a blue that is closer to the wells. The purples should have migrated the same distance from the wells, as the blues should have migrated the same distance from the wells.

Once you have placed your gel into the staining tray bring it to the staining station. Completely cover the gel with methylene blue and cover try with saran wrap. Stain overnight.

Next day--Viewing the gel: Pour the methylene blue back into the bottle and carefully place the gel onto a white light box. The gel is very fragile so take care to not break it. Draw a picture of your stained gel:

Here is a sample gel, with lane "X" containing the DNA from Fluffy's bowl.. Students need to identify which DNA bands come from Fluffy and which bands are the thief's.

What can your data tell you about which cat stole the food?

Students should have an answer based on their data (e.g. I think it's cat #2 because the DNA pieces line up with the DNA pieces from Fluffy's bowl that aren't Fluffy's; The data aren't clear, we can't line up the DNA pieces from the bowl with any of the suspects, we would have to redo the experiment to be sure.). If you're dying to know who stole the food it is cat suspect 2. But, no one tells a jury whether a murderer is guilty, the jurors have to look at the data and decide. So feel free to NEVER tell your students the 'answer', they have to be confident about what their data

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BIOTECH Project Department of Molecular and Cellular Biology The University of Arizona September 19, 2000 Last Modified March 1, 2002 Nadja Anderson, Ph.D. nadja@email.arizona.edu http://biotech.biology.arizona.edu