What Makes Candies So Colorful? Investigate How Gel Electrophoresis Unlocks the Color Code!
|Time Required||Short (2-5 days)|
|Material Availability||This project requires a Bio-Rad kit. See the Materials and Equipment list for details.|
|Cost||High ($100 - $150)|
|Safety||Use caution when heating the agarose. Never connect more than five 9-volt (V) batteries together for this project.|
AbstractImagine that you want to create your own brand of colored candies. You know they would be a success if only you could figure out how to make a certain color, such as purple. How can you do this when government regulators have approved just a few food dyes? First, you will need to find out how your competitors make all of their colors from these few dyes. Do different candy brands use the same or different dyes? This can be figured out with gel electrophoresis, a technique commonly used in laboratories to investigate DNA, proteins, and other molecules. In this science project you will use a kitchen-science version of gel electrophoresis to investigate how candies can have such different colors.
Use gel electrophoresis to investigate food dyes used in candies.
Teisha Rowland, PhD, Science Buddies
This science project was adapted from Bio-Rad's Candy Caper Kit.
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Last edit date: 2017-11-06
Ever wanted to create your own brand of colored candies? One of the first things you will want to do is figure out how your competitors make all of their colors using the few approved food dyes. Do all candy companies use the same food dyes to make the same colors, or do they use different dyes? (To find out which food dyes are approved in the United States, see the U.S. Food and Drug Administration reference in the Bibliography.)
How can you figure out which dyes are used in differently colored candies? By using gel electrophoresis, a technique that scientists apply in laboratories all the time to investigate DNA, RNA, proteins, and other macromolecules and molecules, including food dyes. It is a powerful technique with a wide range of applications. For example, because it can be used to analyze DNA samples, forensic scientists working with police detectives employ it to determine who is a possible suspect based on a blood sample from a crime scene. A forensic scientist can use an enzyme to cut the DNA in the sample at a particular spot, and this creates pieces of DNA unique in size and number to one individual. The scientist can then use gel electrophoresis to "see" the pattern made by these DNA pieces to figure out if two DNA samples match, meaning they both came from the same person.
So how does gel electrophoresis enable a person to investigate and see macromolecules—"large" molecules such as DNA, RNA, and proteins—and molecules, such as food dyes? During gel electrophoresis, samples of macromolecules (or molecules) are put into a gel and then an electrical current is applied to the gel (and to the macromolecules in it). This causes the macromolecules to become separated in the gel, based on their mass and electrical charge. For example, DNA and RNA are negatively charged, so when they are in an electric field, they move away from the negative end and toward the positive end. The gel is usually made of agarose and has many microscopic holes. This causes small macromolecules to travel through the gel faster than large ones, which get stuck more as they move through the gel.
Gel electrophoresis using proteins is similar to gel electrophoresis using DNA, except that proteins are not always negatively charged. But they need to be negatively charged to move through the gel, so how do scientists solve this problem? The proteins are forced to denature, or unfold, in the presence of a chemical that coats them in negative charges. This is how proteins can be separated based on their size using gel electrophoresis.
The equipment for gel electrophoresis is fairly simple. A chamber (often called a "gel box") holds the actual gel. The samples are loaded into the gel in "wells," small holes made on one end of the gel. There is a positive electrode on one end of the chamber, and a negative electrode on the opposite end of the chamber. When the electrodes are connected to a power source, an electric field is created in the chamber. Because the gel is immersed in a buffer solution, it can use the ions in the buffer to provide the current for the gel. Figure 1 shows the gel electrophoresis setup you will use in this science project.
Figure 1. This picture of the gel electrophoresis setup you will create in this science project shows how the agarose gel inside of a gel box has a negative electrode at the top and a positive electrode at the bottom (both electrodes made of paperclips). The power source will be five 9-volt (V) batteries, connected to the electrodes using black and red alligator clips. Each well will contain a color sample.
Aside from the forensics crime-scene example, gel electrophoresis has many other uses. Gel electrophoresis can help determine how many different macromolecules are in a sample of many macromolecules. The size of a macromolecule can be determined by using a sample that is a mixture of molecules of known sizes, called a ladder, in the same gel. By looking at how far the macromolecule travels in comparison to the molecules in the ladder, you can determine the approximate size of the macromolecule. A single type of macromolecule can even be purified by using gel electrophoresis; because the macromolecule is "trapped" in the gel, the region of the gel containing the macromolecule can be cut out and then other techniques can be used to separate the gel from the macromolecule. It can then be purified and used for further experimentation. Watch the video to see how scientists perform gel electrophoresis in a research laboratory. Note: The differences between how you will undertake gel electrophoresis in this project and how it is shown in the video are numerous. This is because you will be performing a kitchen-friendly version of gel electrophoresis without the expensive laboratory equipment. But the scientific concepts are identical.
As mentioned earlier, gel electrophoresis can also be used to figure out which food dyes are in differently colored candies. Food dyes are made up of dye molecules. These are what make some candies, like the M&M's® and Skittles®, shown in Figure 2, so colorful. These different dye molecules can be separated using gel electrophoresis based on the sizes of the molecules. Only a few synthetic dyes are certified and approved for use in the United States by the Federal Food, Drug, and Cosmetic Act (or FD&C), and all of these dyes are negatively charged, like DNA and RNA, so in an electric field they will move away from the negative end and toward the positive end. You can find these dyes in the ingredients list on the packaging of products that use them, often listed as "FD&C" followed by a color and number, such as "FD&C Blue No. 1," or sometimes just listed as "blue 1."
Figure 2. Food dyes make candies, such as the M&M's® and Skittles® pictured here, brilliantly colorful.
In this science project, you will use gel electrophoresis to investigate the food dyes used in candy. How many different types of dye molecules are used to make a certain candy, such as an M&M®, a certain color, such as brown? Is only one type of dye molecule necessary to color it brown, or are there multiple types? Do different types of candy use the same types of dye molecules to get the same color, or do they use different types? You might be surprised by the results!
Terms and Concepts
- Gel electrophoresis
- Electrical current
- Food dyes
- What is gel electrophoresis?
- What are the components of a gel electrophoresis chamber?
- For what kinds of investigations can scientists use gel electrophoresis?
- Which FD&C dyes do you expect are used to make a green M&M®? What about a brown one? Hint: Look at the ingredients on the packaging.
- The University of Utah. (n.d.). Gel electrophoresis virtual lab. Learn. Genetics. Genetic Science Learning Center. Retrieved July 25, 2012, from http://learn.genetics.utah.edu/content/labs/gel/
- DNA Learning Center. (n.d.). Biology animation library: Gel electrophoresis. Cold Spring Harbor Laboratory. Retrieved July 25, 2012, from http://www.dnalc.org/resources/animations/gelelectrophoresis.html
- Bowen, R.A. (2000, January 15). Gel electrophoresis of DNA and RNA. Colorado State University. Retrieved July 25, 2012, from http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/index.html
- LabTricks.com.. (2010, April 12). How to make and run an agarose gel (DNA electrophoresis). Retrieved October 23, 2012, from http://www.labtricks.com/2010/04/12/how-to-make-an-agarose-gel/
- U. S. Food and Drug Administration. (2012, May 31). Food: Food ingredients and colors. Retrieved October 23, 2012, from http://www.fda.gov/food/foodingredientspackaging/ucm094211.htm
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Materials and Equipment
- The Candy Caper Kit from Bio-Rad can be purchased from Amazon.
- Metric ruler
- Permanent marker
- Distilled water (at least 250 mL)
- Measuring teaspoon (1/4); available at Amazon
- Liquid measuring cup, microwave safe (e.g. Pyrex) (at least 250 mL); available at Amazon
- Large glass jar that can hold at least 250 mL (approximately 1 cup). Make sure it is clean. Alternatively, you can use a bowl or any other container, although one with a leak-proof lid is easiest.
- Wax paper (2 sheets, each large enough to easily cover the entire top of the provided beaker)
- Microwave oven
- Oven mitts
- Plastic ruler, plastic knife, or butter knife
- M&M's® and Skittles® candies:
- Brown, yellow, blue, red, and green candies (3 candies of each color)
- Any additional colors you want to test (3 candies of each color)
- Include some M&M's® and Skittles® that are the same, or a similar, color. For example, a red M&M® and red Skittle®.
- You can test up to eight different candy colors/types at once, and a total of 32 colors/types with the kit if you run multiple trials.
- Optional: additional plastic cups (the Bio-Rad kit provides 5 cups)
- 9-V batteries, new or fully charged (3-10 depending on number of gels and running speed of each gel); 3-5 batteries can be used per gel where more batteries means a faster run time (see the Procedure for details).
- Clock or timer
- Optional: camera
- Lab notebook
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Remember Your Display Board Supplies
Poster Making Kit
ArtSkills Trifold with Header
Preparing the Gel Box and Pouring the Agarose Gel
Before starting the project, read through the instruction manual that comes with the Bio-Rad kit. The procedure that you follow in this project will be in a slightly different order from the one described in that manual, so do not be surprised when the procedure here is a little different. To start this project, you will assemble your gel box and pour the agarose gel.
- The hinged plastic box that comes with the Bio-Rad kit will be used as two gel boxes. Carefully separate the box bottom and top so that you have two gel boxes.
- Open the hinged plastic box and remove its contents.
- Gently pull the two halves of the box apart at the hinges, being careful not to crack or break either half. Do not worry if the hinge piece breaks off.
- Use the permanent marker to mark the gel boxes. You will be marking where to put the gel comb and where to cut the gel to insert the electrodes later.
- On one end of the box where the hinges are, draw a large "plus" (+) sign on each half of the box. Mark the other side (without hinges) of each half of the box with a big "minus" (-) sign.
- On the long side of your box, at the end where the hinges are, use the ruler and marker to make a mark 1 centimeter (cm) from the end. Do this on both sides of the hinged end, for each gel box.
- At the other end of the gel boxes (where there are no hinges), make marks at 1 cm and 3 cm from the end on the long side of your box. Again, do this on both sides of the end with no hinges, for each gel box.
- You should now have six marks on each half of your box, three on each long side.
- Using the 10x multipurpose solution that comes with the Bio-Rad kit, make up 250 mL of 1x multipurpose solution with distilled water.
- Pour all of the 10x multipurpose solution into a measuring cup.
- Then add distilled water to the cup until it reaches the 250 mL line.
- Transfer the solution into a jar with a lid. Put the lid on tightly and shake the solution to mix it.
- You now have a jar with 250 mL of 1x multipurpose solution.
- Prepare the agarose solution using the 1x multipurpose solution. To make the agarose solution, use 1/4 teaspoon of agarose (included in the Bio-Rad kit) for every 75 mL of 1x multipurpose solution. You will make two agarose gels.
- Add 75 mL of 1x multipurpose solution into your beaker and add 1/4 teaspoon of agarose/agar to the solution.
- Swirl the beaker slightly to mix and then place the container in the microwave oven to dissolve the agarose.
- Put a sheet of wax paper over the beaker, covering the entire top. Fold the edge of the wax paper over the edge of the beaker slightly, to keep the wax paper in place.
- Microwave the solution on high for about 30–35 seconds.
- Put on oven mitts and slowly swirl the solution between your hands, being careful not to spill any of it.
- Continue microwaving the solution, stopping it every 20 to 30 seconds to swirl. If you see the solution bubbling, stop it and swirl it, or it may overflow.
- Stop microwaving the solution when all of the small transparent agarose particles have dissolved. You will probably need to microwave the solution for anywhere from one and a half to three minutes before all of the agarose has dissolved.
- Tip: The agarose solution may have lost a little liquid due to evaporation during microwaving. Because the beaker was covered with wax paper, very little evaporation should have occurred, and this should not be a concern. However, if it looks like the agarose solution lost significant volume, you can pour distilled water into the bowl until it is at its original volume (75 mL) and try to re-microwave the agarose solution.
- In one gel box, put the 6-well foam comb (included in the Bio-Rad kit) between the 1 cm and 3 cm marks you made on the end with the "-" sign in step 2.
- Make sure the comb is standing straight up, with the teeth facing down. Center the teeth of the comb so that they are not touching the box.
- Your box should now look like Figure 3.
Figure 3. In the gel box, place the comb so that it is centered between the 1 cm and 3 cm marks (seen as black dots on the right side of the gel box) and the comb teeth are facing straight down.
- Carefully pour all the hot agarose solution into the gel box.
- There should be enough agarose solution in the box so that it rises part of the way up the comb's teeth.
- The more the teeth are covered with solution, the deeper the wells will be, and the larger amount of sample you will be able to put in the gel. However, if the gel is too thick, it may be hard to see your samples.
- There should be enough agarose solution in the box so that it rises part of the way up the comb's teeth.
- Wait for the gel to solidify. This will take about 10 to 20 minutes at room temperature.
- Tip: When the gel has solidified, it should be firm to the touch and a little cloudy, or a little opaque.
- When you are sure that the gel is completely set, gently pull the comb up and out of the gel. The wells that were made by the comb will serve as reservoirs for your samples.
- Once you removed the comb, repeat steps 4–8 to prepare your other gel using the second gel box.
- At the 1 cm marks you made on both ends of the box halves, use a plastic ruler, plastic knife, or butter knife to cut a slab off both ends of the gels. The cut should go from the 1 cm mark on one side to the same mark on the other side.
- Press directly down with the ruler or knife, going all the way through the gel and to the bottom of the box.
- Do not slice the gel, but pierce it multiple times across the width of the gel.
- Carefully lift the slab out and discard it.
- When you are finished, both gel boxes should be missing a 1 cm strip of gel at opposite ends.
- Add enough 1x multipurpose solution to each gel box to just barely submerge the gel. You should be able to see slight indentations in the surface of the 1x multipurpose solution on the top of the gel where some of the wells are.
- If there is too much 1x multipurpose solution in the gel boxes, this could make it harder to load your samples into the gel. If so, pour the extra solution back into your 1x multipurpose solution container.
- Both ends, where you cut the gel, should be full of solution, too. If there is not enough 1x multipurpose solution, the electric current will not be able to go through the entire gel. Add more buffer to make sure to completely submerge the gel.
Extracting Dyes from Candies
In this part of the science project you will extract dye from the candies you have selected to test.
- Decide on and select the colors of M&M's® and Skittles® candies you want to test.
- Include brown, yellow, blue, red, and green M&M's® or Skittles®, in addition to whatever other colors you want to test.
- For some of the colors you test, include both M&M's® and Skittles® of that color. For example, maybe test both green M&M's® and green Skittles®.
- You can test at least ten different candy colors.
- For each candy type and color you want to test, pick three. For example, if you want to test green M&M's®, pick three green M&M's®.
- Using the permanent marker, label plastic cups with the candy types and colors you selected.
- Fill the provided pipet about 1/3 of the way up the large bulb with 1x multipurpose solution and add it to one of the cups. Repeat this process for each of your cups until you have 1x multipurpose solution in all of the cups.
- Place three of each kind of candy (which you picked in step 1d) into the appropriate cup.
- Swirl each cup a little.
- Make sure that all the candy pieces get some solution on them.
- Swirl each cup multiple times until it looks like the color coating has dissolved off of the candies and you mostly see the white or brown layer below it. The cups can sit momentarily between each time you swirl them.
- Once the color coating has dissolved, remove the candies from the solution with a clean spoon.
Agarose Gel Electrophoresis
In this part of the science project, you will run your extracted candy dyes on the gel electrophoresis apparatus you constructed. You will run these samples with a reference dye solution to help you figure out what dyes are in your samples.
- Carefully unfold four paperclips that came with the Bio-Rad kit so that each paper clip has a long flat section and a short piece sticking straight up on either end (one end piece will be slightly longer than the other). See Figure 4 for how to do this.
- Make the long section as flat as possible.
- These paperclips will be your electrodes.
Figure 4. Unfold the paperclips until each paperclip has a long flat section in the middle and a short piece sticking straight up on either end (one end piece will be slightly longer than the other).
- Place the flattened paperclips inside the gel boxes, with the end pieces sticking straight up. Put one on either end of each box.
- Place them as far from the gel as possible (close to the ends of the box).
- The slightly longer ends of the paperclips should be on the same side in each gel box.
- Tap the tube with the FD&C dye mix (which came with the kit) firmly on a hard surface.
- This should help remove dye from the inside part of the lid of the tube, knocking it down into the rest of the dye. Keep tapping the tube until the lid has very little, or no, dye on it.
- In your lab notebook, make a table that shows the order in which you will load your samples into the gel, similar to Table 1. Include rows to record your observations.
- Make a separate table for each gel.
- For each gel you will load the FD&C dye mix solution and five samples.
- The dye mix solution contains the four most commonly used food dyes: Blue 1, Yellow 5, Yellow 6, and Red 40. You will be comparing the results using the reference dyes with the results using your samples. Doing this you should be able to figure out which of these common food dyes are used in your samples.
|Dye||FD&C dye mix||Sample 1||Sample 2||Sample 3||Sample 4||Sample 5|
|Number of bands|
|Migration distance of each band (cm)|
|Color of the bands|
- Using the transfer pipet, load your samples into each gel according to the table in your lab notebook.
- Well #1 should be on the far left, while well #6 should be on the far right.
- Fill only the neck of the transfer pipet with your dye solution to only load a very small amount of dye into each well.
- While filling the pipet hold it slightly compressed so that you do not suck up any air, and then squeeze out less than a drop into the well.
- If you release too much dye, it may leak into nearby wells and contaminate them with the wrong dye. If you do not release enough dye, your results may look faint.
- If you have any extra dye, squeeze it back out into the container it came from.
- If some dye leaks into a nearby well and contaminates it, make a note of this in your lab notebook.
- Tip: You can place a piece of dark paper underneath the gel box to make the individual wells more visible.
- Fill a cup full with distilled water and between loading different samples rinse your pipet out in the water, sucking in and squirting out water several times until the dropper looks clean.
- If you do not do this, you might contaminate wells with the wrong dye.
- Connect your 9-V batteries together in series by snapping the positive (+) terminal of one into the negative (-) terminal of another until you have formed a battery pack with all three or five batteries, as shown in Figure 5. There should be one positive and one negative terminal left exposed.
- You can run one gel with three to five batteries. Using three batteries should take at least 20 minutes to run. Using five batteries should take about 15 to 20 minutes.
- Note: Never use more than five batteries to run one gel. The apparatus is designed to work only at low voltages (≤45 volts; 45 volts is supplied by five 9-V batteries).
- If you do not want to wait until your first gel is done to start running the second gel, use 6 batteries total and run one gel with three batteries and the second with another three batteries.
Figure 5. Form a battery pack using all three or five 9 V batteries, as shown here.
- Using the alligator clip leads, attach the battery pack to the paperclips (electrodes) in the gel box. The negative terminal of the battery pack should be connected to the electrode at the end of the gel box nearest the wells, that you marked with "-", and the positive terminal should be connected to the electrode farthest from the wells on the side that you marked with "+"; you want the dye to migrate towards the positive terminal as the dye separates.
- Take the black alligator clip cable and connect one end to the paper clip electrode on the side that you marked with a "-".
- Then attach one end of the red alligator clip cable to the electrode on the side of the gel box that you marked with a "+".
- To start the gel electrophoresis, connect the electrodes to the battery pack by connecting the other end of the black alligator clip cable to the "-" side of the battery tower, and the free end of the red alligator clip cable to the "+" side of the battery pack. When it is all connected, it should look similar to Figure 6, below.
- As you connect the electrodes to the battery pack, make sure the flattened part of the electrodes stays submerged in the buffer, on the bottom of the gel box, with the ends of the electrodes above the buffer surface. Keep the electrodes as far away from the gel as you can.
- It may take some patience to keep the electrodes positioned so that they do not fall over when connected to the alligator clip leads.
- When the battery pack and electrodes are all properly connected, you should see bubbles forming around the electrodes in the solution.
- If you do not see bubbles, recheck all your electrical connections. Make sure the batteries are properly placed in series, and that they are fresh and fully charged.
Figure 6. When your gel electrophoresis setup is all connected and ready to run, it should look similar to this picture.
- Check on your gel in 15 to 20 minutes. Once you see good migration and separation of the dyes, you can stop the gel by disconnecting the alligator clip cables from the battery pack.
- Note: For some of the dyes you should be able to see the dye separate into multiple distinct bands of color that make it up. Check that you see this before stopping the gel.
- Look at the results on your gel and fill out the data table in your lab notebook.
- The same dye in different samples should have the same migration distance of each band (as measured from the well to the band) and have bands of the same color. Knowing this and knowing what dyes are in the reference dyes, which dyes are used for each colored candy?
- Do some candies have multiple dyes? If so, do the different dyes they have make sense? Are you surprised by any of your results?
- Did the manufacturers of M&M's® and Skittles® use the same dyes to make the same colors?
- Which dyes do you think may be the largest, and which may be the smallest? Hint: Re-read the Introduction.
- If you see a dye for a candy that does not look like it matches any of the reference dyes (it is a different size and/or color), look at the ingredients on the packaging for that candy and think about what other dye it might be.
- If you have a camera, take a picture of your gel.
- Once you have finished running your first gel, repeat steps 3-9 with the second gel.
- If your electrophoresis chamber does not work as well as it did the first time, try replacing the batteries with fresh, fully charged ones. Running the electrophoresis chamber can drain the batteries.
- If you want to test more than ten samples, you can repeat this entire project as long as your Bio-Rad kit supplies last.
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- What dyes do other kinds of candies use? Repeat this science project using candies other than M&M's® and Skittles® (such as Runts®, Reese's Pieces®, jawbreakers, Red Hots®, Hot Tamales®, or Mike and Ike®). Do all candies use the same dyes for the same colors, or do they use different dyes? Do any candies use dyes that are not among the four reference dyes in the Bio-Rad kit? How often does a candy use at least two different dyes?
- Food dyes are added to many foods other than candies. Some other foods you could test with this science project include Kool-Aid® drink mixes and JELL-O® dessert mixes. Repeat this science project using these mixes (dissolve each in a little bit of distilled water). Which food dyes are used in the drink mixes? Do your results match the dyes listed on the packaging? If multiple dyes are used in a drink mix, is more of one of the dyes used than the other(s)?
- What variables affect the rate of electrophoresis separation? There are many variables to explore. Several to start with might be the type of electrode, the amount of power, and the agar percentage of the gel.
- Use your gel electrophoresis to determine if two different types of plants use the same molecules for pigment. To prepare your samples, take the flowers from each plant, grind them up, add a little bit of isopropyl alcohol, and continue grinding. Once the solids settle, pour the pigment-tinted alcohol from each flower into a separate container. Let most of the alcohol evaporate and then add a drop or two of your 1x TAE buffer solution to reconstitute the pigments from each flower.
- How does the composition of the gel affect how your samples run? Make an agarose gel that is greater than 1%, and one that is less than 1%, then compare how the same samples run on the gels.
- In this science project you used agarose in your gel electrophoresis, but can substances other than agarose, such as gelatin, also be used in gel electrophoresis? Try this project again using a gel made of 10% gelatin (12 grams of gelatin with 120 mL of 1x TAE buffer). How is doing the gel electrophoresis using gelatin different than using agarose? Does one seem to work better than the other? What other substances might work as well, or better than, gelatin and agarose in making a gel for gel electrophoresis?
- The pH of a solution depends on how many hydrogen ions are present. You can do this science project and carefully measure the pH (using pH paper) near the positive electrode and the negative electrode while the gel is running. What is the pH at both ends? What does this say about the number of hydrogen ions at both ends? Can you make sense of your results?
- This project uses a gel electrophoresis kit to explore dyes in foods, but you can do many other projects easily at home that explore gel electrophoresis or DNA, which is often investigated using gel electrophoresis. Here are some additional related Science Buddies projects:
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