Bubble gum Mystery

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'. You can use whatever crime scene you would like to set up. The gum scenario is designed to show a match of DNA fragments for one of the suspects and the crime scene (unless we have ABC gum…) You also have the option of having the DNA from the crime scene a mixture of the victim and a suspect (i.e. the cat food caper.) The match is much easier to read, and I prefer to save the mix for a paternity case such as the whale paternity activity. However you can make up any scenario that you wish, just be certain that the DNA you have makes sense for your scenario.

The Scenario:

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 solve the crime?

   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 match the pieces at the crime scene.

4. What tricks can we use to see DNA?

   DNA stain such as methylene blue

Materials                   (for each group)

DNA from suspects (#1 and 2, and #3), and the crime scene (X)

1 micropipette

4 tips

plastic dish

electrophoresis apparatus

pair red and black wires

power supply

(for the class)

agarose (boiled in a microwave and kept in a 55°C water bath)

Tris-acetate/EDTA solution (TAE)

methylene blue

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.  Why near the black electrode? 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.

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. 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. Gently remove the stoppers and comb and load your DNA samples 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. Now gently pour TAE solution over your gel so that is it 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 f or 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 blue 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.

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

What can your data tell you about who committed the crime?

Students should have an answer based on their data (e.g. I think it's suspect #2 because the crime scene DNA pieces line up with suspect #2's DNA pieces; The data aren't clear, we can't line up the crime scene DNA pieces with any of the potential suspects, we would have to redo the experiment to be sure.). If you're dying to know whose DNA is at the crime scene, it's #2 and 3. 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 show.

Recipes and Background Information

• DNA samples

For 10 reactions each:

>>>>

Sample #1	Sample #2	Sample #3	Sample #4
65 µl water	65 µl water	65 µl water	20 µl water
20 µl lambda DNA	20 µl lambda DNA	20 µl lambda DNA	20 µl lambda DNA
10 µl buffer #3	10 µl buffer #2	10 µl buffer #2	5 µl buffer #2
5 µl EcoRI	5 µl PstI	5 µl HindIII	5 µl PstI
			20 µl water
			20 µl lambda DNA
			5 µl buffer #2
			5 µl HindIII

Incubate at 37°C for at least 2 hours, and up to 12 hours. For samples #1-3, add 20 µl 6X loading dye and pipet into 12 µl aliquots. For the 'X' sample, combine two reactions, add 20 µl 6X loading dye, and pipet into 12 µl aliquots.

• 6X loading dye

6 ml 50% glycerol

1 ml 2% bromophenol blue

1 ml 2% xylene cyanol

2 ml distilled water

• Tris-acetate-EDTA buffer for dye electrophoresis

>

10X stock (per liter)

48.4 g Tris base

11.4 ml glacial acetic acid

20 ml 0.5M EDTA (pH 8.0)

Add distilled water to make total volume 1 liter. Dilute to make 1X working solution (100ml stock, 900 ml distilled water).

• 0.8% Agarose gel for dye electrophoresis

In 250 ml Pyrex bottle, combine:

125 ml 1X TAE

1 g agarose

Microwave uncovered to melt agarose, be careful not to boil over on microwave or your hand. Cover loosely and store at room temperature.

• 0.025% Methylene blue stain

Make 1% methylene blue stock: 99 ml water and 1 g methylene blue powder.

10 ml 1% methylene blue stock

390 ml distilled water

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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