A. Introduction. Figure 1 Figure 2

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1 Varsity Biology Molecular Biotechnology Lab Decode the Candy Dye Essential Questions: How can we use Gel electrophoresis to separate molecules? How do we use the equipment (which will be important in the labs coming up!) A. Introduction 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 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. 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 below shows the gel electrophoresis setup you will use in this science project. Figure 1 Figure 2 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. 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. In this investigation you will use gel electrophoresis to investigate the food dyes used in candy. You will be given a vial of an unknown dye combination that is the product of several different dyes from several different candies. You will figure out what its dye components are. By doing so you will learn how to use the components of a gel electrophoresis system.

2 Terms and Concepts Dye Extraction From Candies Gel electrophoresis Electrical current Food dyes Student Workstation Quantity DNA Micropipettor Electrode Agarose Dye extraction solution Macromolecules Charge 2 ml 2 ml microcentrifuge tubes 4 B. Methods and Microcentrifuge Materials tube rack 1 Marking pen 1 Each student workstation: Plastic cups or small beakers Quantity 4 Colored candies 4 varieties, 1-4 candies per variety Eyedropper 1 Dye extraction solution 2 ml 2 ml microcentrifuge Colored tubes candies (empty) 4 4 varieties, 1 4 candies per variety* Microcentrifuge tube containing unknown dye 1 1 ml Pipette and *Candy pipetter example: 3 green Skittles, 3 orange 1 jelly of each beans, 4 Red Hots, 1 brown gumball Glass vials 4 Protocol Agarose gel chamber 1 Agarose gel tray 1 Blue Tape 1 1. Label the four microcentrifuge tubes with your Agarose gel (prepared using instructions below) 1 initials and the names and colors of the candies Power supply (shared with other groups) 1 you are using. Electrophoresis buffer, 1x TAE (wait until needed) 275mL 2-20 L adjustable-volume micropipette 1 Micropipette tips 5 Marking pen 1 2. Label four cups with your initials and the names and colors of the candies you are using. C. Protocol (Divide your lab group and two students work on Part 1 and two students work on Part 2) Day 1 / Part 1: Extract your dyes: 1. Work with the class to divide up the possible candy dye sources so that all dye sources will be tested. You 3. Using an eyedropper or pipet, add 0.5 ml of will only make dye four extraction dye extractions solution but to each there cup. are many Use the other colors and candies. When all the groups pool their data you will be able to get data for all colors of the Skittles, M&Ms, and Jellybeans. volume marks on the 2 ml microcentrifuge tube to measure the correct volume. 2. Label the four microcentrifuge tubes with your initials and the names and colors of the candies you are using. 4. Place your candy into the appropriately labeled cup and swirl the candy in the dye extraction solution. If using a candy such as M&M S or 3. Using a pipet, add 0.5 ml of dye extraction solution to each vial. 4. Place your candy Skittles, into just a vial dissolve and swirl the color the candy coating the off until dye extraction solution. If using you a get candy to the such white as layer M&M S of the or Skittles, candy. For just all dissolve the color coating other off until candies, you get try to to the get white as dark layer a solution of the candy. of For all other candies, dye try as to possible. get as dark a solution of dye as possible. 5. Remove your candy from the vial. Pour the solution containing the dissolved 5. colored Remove candy your coating candy into from the the appropriately cup. Pour the labeled microcentrifuge solution tube. containing The dye extraction the dissolved vials colored can be stored overnight. candy coating into the appropriately labeled microcentrifuge tube. ******** CLEAN UP YOUR LAB AREA THOROUGHLY *********** 20

3 Day 1 /Part 2: Prepare your gel 6. Seal the ends of the tray securely with strips of blue tape. Press the tape firmly to the edges of the gel tray to form a fluid-tight seal.! 7. Place the comb in the appropriate slot of the gel tray. Gel combs should be placed in the slot nearest the end of the casting tray. The comb will form the wells into which the samples will be loaded. 8. Carry the tray to the hot water bath containing the agarose gel and place the tray on the lab table. 9. Carefully pour the molten agarose solution into the gel tray until it almost completely covers the gel comb teeth or until the gel is cm thick.!! 10. Allow the gel to solidify at room temperature for 10 to 20 min. It will appear cloudy or opaque when ready to use. 11. Carefully remove the comb from the solidified gel by pulling gently in an upward direction. 12. Remove the tape from the edges of the gel tray.! 13. Store the gels in a sealable plastic bag with 1 2 ml of 1x TAE buffer at room temperature for 1 day, or store in the refrigerator (4 C) for up to 1 week before using them. Label your plastic bags. ******** CLEAN UP YOUR LAB AREA THOROUGHLY ***********

4 Day Place the casting tray with the solidified gel in it into the platform in the gel box. The wells should be at the ( ) cathode end of the box, where the black lead is connected. If the comb is still in the tray, remove it very carefully from the gel by pulling it straight up. 15. Fill the electrophoresis chamber with 1x TAE buffer to cover the gel, using approximately 275 ml of buffer. 16. Do no carry out the next step until you have watched the teacher demonstrate proper loading technique. Using a separate tip for each sample, load 10 µl of your four candy dye extracts into the gel, with one sample per well. The first sample is loaded in the well at the left hand corner of the gel. Also load one well with the unknown dye extract. (You have three extra wells in case you make mistakes) Use the gel diagram to write down what is loaded into each well. 17. Place the lid on the electrophoresis chamber. The lid will attach to the base in only one orientation. The red and black jacks on the lid will align with the red and black jacks on the base. Plug the electrodes into the power supply. 18. Turn on the power supply. Set it for 100 V and electrophorese your samples for 15 min. 19. When the electrophoresis is complete, turn off the power and remove the top of the gel box. Carefully remove the gel and tray from the gel box. Be careful the gel is very slippery! 20. Carefully slide the gel out of the tray and onto the mini whiteboard. Take a photograph of the gel for your records immediately. 21. Collaborate with the other groups to identify the dyes present in the unknown

5 D. References: The University of Utah. (n.d.). Gel electrophoresis virtual lab. Learn. Genetics. Genetic Science Learning Center. Retrieved July 25, 2012, from DNA Learning Center. (n.d.). Biology animation library: Gel electrophoresis. Cold Spring Harbor Laboratory. Retrieved July 25, 2012, from Bowen, R.A. (2000, January 15). Gel electrophoresis of DNA and RNA. Colorado State University. Retrieved July 25, 2012, from LabTricks.com.. (2010, April 12). How to make and run an agarose gel (DNA electrophoresis). Retrieved October 23, 2012, from U. S. Food and Drug Administration. (2012, May 31). Food: Food ingredients and colors. Retrieved October 23, 2012, from