Agarose Gel Electrophoresis of Food Coloring Dyes
Agarose gel electrophoresis allows you to separate molecules according to size. It is one of the most important procedures used in studies of DNA. To learn how to do it, we will use agarose gel electrophoresis to show that food color dyes are often made up of several dyes that are different colors.
Before we start, let's think about what we are going to do. First of all what is agarose? It is a polymer! Agarose, a highly purified form of agar, is a polymer that is made entirely of sugar molecules. It is produced by a kind of seaweed and is used to give thicker consistency to foods such as ice cream. We use agarose because a solution of agarose and water forms a gel when it cools to room temperature. It is sort of like jello except that it does not get soft when it gets warm.
Molecules like food dyes can move through an agarose gel, but the larger they are, the slower they move. To understand the process, think about a backyard or woods that is full of trees. If you watch, you can see that small birds fly through the branches of the trees almost as if they were not there. What about a large bird like a hawk or an owl? They can only fly through larger spaces among the branches or they have to fly around the trees. Therefore, they cannot fly as fast as the smaller birds. In the same way, small molecules can move quickly among the agarose branches in the gel, but larger molecules move more slowly because they have to pass through larger spaces.
What is electrophoresis? Electrophoresis is process that uses electricity to pull molecules from one place to another. If you look at the gel box, you will see that it has two bare wires called electrodes. One is connected to a red wire and has a positive charge, and the other is connected to a black wire and has a negative charge. Most dyes have a negative charge so they are attracted by the positive charge and move through the gel towards the positive electrode. Therefore, we are going to use agarose gel electrophoresis to pull dye molecules through an agarose gel and separate them according to size and positive charge.
Food color dyes
1) Weigh out 1.2 grams of agarose and add it to 100 ml of room temperature TB buffer. Swirl to make sure that there are no clumps. Boil the mixture to melt the agarose by heating it in a microwave. As soon as it comes to a boil, open the microwave and swirl the flask without removing it from the microwave. Be sure to handle the hot flask with a glove or hot pad! After swirling, remove the flask and look at the contents. You will see clear particles moving in the solution. These particles are unmelted agarose. Return the flask to the microwave and repeat the boiling and swirling process until you can no longer see the agarose particles. Repeat the boiling and swirling process one more time to be sure all the agarose particles are in solution.
2) Let the agarose solution cool but not too much. It should feel very hot but not so hot that you cannot hold the flask. Pour enough into the gel tray to make a gel that has thickness of about 3 mm. Insert your gel comb into the liquid agarose in the gel tray. Cover the rest of the agarose, let it cool, and save it for your next experiment. (To reuse a solidified agarose solution, simply reheat it with occasional swirling until the solution is uniformly liquid).
3) Once the agarose in your gel tray has solidified, remove the comb. The holes left by the teeth of the comb are called wells. Place the gel tray in the gel box. Add enough TB buffer to the gel box to cover the gel with about 2 mm of buffer.
4) Slowly and carefully transfer 3 microliters of one of the food dyes into one of the wells. Repeat with the other dyes. (Use every other well of the gel.)
5) Place the lid on the gel box, and turn on the power supply to 100 volts.
6) Look at the gel from time to time to see how the dyes are separating from one another. Notice how each dye moves in a straight line from its well towards the positive electrode. Thus, just like swimmers at a swim meet, each dye stays in its own lane. Also, you can see some dyes moving faster than others. Once the fastest dye moves about two thirds of the way through the gel, turn off the power supply and remove the gel tray from the gel box.
7) How many colored spots do you see in each lane? Draw a picture of the gel in you notebook. Each lane contains all of the different colored dyes found in one food color. Make a table showing the different colored dyes that are used to make each of the food colors that you loaded on your gel.
8) Measure how far
the center of each dye spot has moved from the edge of the well and record all
the measurements in your notebook. Based on these observations which color
dyes move the fastest? Which are the slowest? Think of two reasons why would
one dye move faster than another.