Agarose Gel Electrophoresis with Dyes - Teacher Guide

What is electrophoresis? In this activity, students use agarose gel electrophoresis to determine the composition of different biological dyes. Students learn how molecules can be separated and identified by electrophoresis. This activity is a good introduction to electrophoresis, and we like to do this lab before working with gel electrophoresis of DNA.

Materials and Equipment

Protocol

1. Get your electrophoresis apparatus. Make sure the comb is in place (there should be 1 comb in the middle) 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, this takes 5-10 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 pour TAE solution over your gel so that it 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

4. Gently remove the comb and use a pipet to load each dye into a separate well in gel. Use a separate pipet tip for each sample. Draw a diagram on the next page as you load the dyes to help you keep track of which samples you put where!

Diagram of gel: 

Some of 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 dye samples. This drawing will be useful during the analysis, once the dyes are separated.

5. 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.

6. Electrophorese samples for ~10 minutes. Turn off power supply, disconnect power cords from the chamber, and remove top of electrophoresis chamber.

Analyzing your dye data

1. Carefully remove casting tray with gel and draw a picture of your gel using colors.

2. Measure distance traveled by the bands in the dyes. Measurements are typically done from the leading edge of the well to the leading edge of the dye band. Also record whether the bands traveled toward the positive or negative pole.

Dyes move to the positive electrode if they are negatively charged, and to negative electrode if they are positively charged (the old adage 'opposites attract'). They separate because they are different sizes, dyes that move faster and farther from the wells are smaller (lower molecular weight).

3. Compare known dyes with the unknowns. Which dyes are present in the unknown?

Students should be able to figure this out based on the pattern of dyes after electrophoresis, but if you're itching to know, it's: Bromophenol blue, Safronin O, and Xylene Cyanol.

Classroom time needed for this lab


• 50 minutes (if agarose gels are poured before class)
• 90 minutes (if students pour the agarose gels)

You will need the following materials:


• 1 x TAE
• Agarose
• Various dye mixtures: Methyl Orange, Bromophenol Blue, Xylene Cyanol, Pyronin Y, Safranin O, Unknown
• Practice Loading dye

If you do not have a variety of biological dyes available, you can prepare a simpler version of this experiment that uses food color. Refer to the lab activity, "Agarose Gel Electrophoresis with Food Color."

You will need the following equipment and supplies:


• Micropipets and tips to load dye samples
• Small microcentrifuge tubes (0.5 ml or 0.65 ml size)
• Electrophoresis units and power supplies
• 1X TAE for electrophoresis units
• Microwave oven
• Hot water bath for keeping agarose liquified

Setting up the classroom for this lab

Distribute at each lab station (we use four students per group):

• 12 ul of each dye in small, labeled microcentrifuge tubes (6 tubes total)
• 4 small microcentrifuge tubes each containing 12 ul practice dye
• Micropipet and tips
• Electrophoresis unit and power supply

We use a central station for agarose and 1X TAE. We use a hot water bath to hold four small bottles, each containing 125 ml of 0.8% agarose. The high temperature keeps the agarose liquified. We have four large bottles at room temperature, each containing 1000 ml of 1X TAE.

If necessary, demonstrate to students how to use the micropipet. While the agarose is solidifying in the gel tray, the students can practice using the micropipet with the practice dye. After the gel is solidified and 1X TAE is added to the electrophoresis unit, students can practice loading the gels with the practice samples. Important: Practice samples are loaded at the positive (red) end of the gel, and the dye samples are loaded in the middle set of wells.

The 1X TAE in the electrophoresis units can be poured back into the large bottles for reuse, and the used gels can be disposed in the regular trash or taken home by the students. If the students want the gels, wrap them in plastic wrap. Unfortunately, the dyes in the gel diffuse rapidly, so the banding patterns will disappear overnight.

Other notes:

1. We use the Graduate Micropipets available from Life Technologies, and we use regular yellow tips (1-200 ul). The Graduate micropipets are much less expensive than micropipets commonly found in research laboratories, and they are more likely to survive in student hands.
   
   
2. Be sure to calculate how much agarose you will need for the lab. You can add a defined volume of water into the gel tray to determine the volume of agarose that would be needed for each tray.
   
   
3. Be sure to calculate how much 1X TAE you will need for the lab. Using a graduated cylinder, you can pour a defined volume of water into the electrophoresis unit to determine the volume of 1X TAE that would be needed for each unit.
   
   
4. If there is not enough time during the class for students to pour the gels and run the electrophoresis units, you can pour the gels before class. After the gel solidify, they can be stored in the refrigerator in a ziplock plastic with some 1X TAE buffer.

Recipes

TAE is the buffer used in gel electrophoesis.

1. Put 10.8 g Tris base, 5.5 g boric acid, and 0.74 g EDTA into a 1000 ml beaker.
   
   
2. Fill a graduated cylinder with 1000 ml of deionized or distilled water. Add the water to the 1000 ml beaker containing the dry chemicals.
   
   
3. Put beaker on magnetic stirrer, and add stir bar to solution. Keep stirring until chemicals are dissolved. This solution is 1X TAE.
   
   
4. Store 1X TAE buffer solution in a large container. The 1X TAE can be kept indefinitely at room temperature. This buffer can be reused several times before disposing down the sink.
   
   

Agarose is the gel matrix used to separate molecules, such as DNA and dyes, during electrophoresis.

1. Weigh 1 gram of agarose on a folded piece of weighing paper and add to empty 250 ml bottle or flask.
   
   
2. Add 125 ml of 1X TAE buffer solution to agarose. Note: The container should never be filled more than half-way in order to prevent the solution from boiling over.
   
   
3. If a bottle is used, cap the bottle loosely to release air during boiling. If you using a flask, cover the opening and neck of flask with plastic wrap.
   
   
4. Mix solution by swirling. Microwave the agarose solution at high heat until powder is completely dissolved. The length of time required will vary depending on the microwave oven. The molten agarose solution should look clear.
   
   
5. To keep the agarose liquified (for example, during several biology classes), store the bottle or flask of agarose in a hot water bath between 600 and 700C. Be sure that the bottle or flask is covered to prevent evaporation. A hot plate with a pot of water can substitute for a laboratory water bath.
   
   
6. If there is agarose left over in the container, you can let the agarose solidify and store it at room temperature until next use. Be sure that the container is well covered
   
   

This procedure requires making a 2% stock for each dye. The dye stocks are then diluted into a sugar-based solution for loading the agarose gel. The sugar, such as glycerol, increases the density of the sample so that it sinks to the bottom of the well.

The dye stocks are used to prepare samples for agarose gel electrophoresis with dyes.

1. Measure 0.2 g of one of the dye powders on a square piece of folded weighing paper.
   
   
2. Using the crease in the paper, pour the dye powder into a tube. The tube should be able to hold between 10 and 15 ml.
   
   
3. Add 10 ml deionized or distilled water to the dye and cap the tube. Shake the tube well to dissolve the dye in the water. For some dyes, not all the powder will go into solution.
   
   
4. Repeat steps 1-3 for each dye.
   
   
5. The dye stocks (2% concentration) can be stored indefinitely at room temperature.

The dye samples for electrophoresis are prepared from 2% dye stocks. The colored samples allow students to observe the dye molecules migrate during electrophoresis. One of the samples is the "unknown," which contains a mixture of three of the other dyes.

The dye stocks are Methyl orange, Bromophenol blue, Xylene cyanol, Pyronin Y, Safranin O, and Unknown.

1. Prepare a 50% glycerol solution. Pour 10 ml glycerol into a 25 ml graduated cylinder (note: glycerol is very viscous). Next, add 10 ml deionized or distilled water to the same cylinder. Use a stirring rod (a pipet would also work) to mix the glycerol and water. The glycerol solution can be stored in a small, closed container in the refrigerator.
   
   
2. Prepare a dilution of the dye stock for electrophoresis. Using a pipet, add 2 ml 50% glycerol solution into a 25 ml graduated cylinder.
   
   
3. In the same cylinder add 1 ml of a 2% dye solution.
   
   
4. Add 17 ml deionized or distilled water to make a final volume of 20 ml. Use a stirring rod to mix the ingredients together. This batch of dye sample is ready to use - we use 12 ul of the dye sample per lane on an agarose gel. You can store any unused dye sample in a small closed container in the refrigerator. Repeat steps 2 - 4 for each dye stock.
   
   
5. Prepare the unknown dye sample. Add 2 ml 50% glycerol solution to a 25 ml graduated cylinder. Next, add 1 ml each of 2% methyl orange, 2% bromophenol blue, and 2% xylene cyanol. In the same cylinder add 15 ml deionized or distilled water. Use a stirring rod to mix the ingredients together. The unknown dye sample is ready to use, and store any unused dye sample in a small closed container in the refrigerator.
   
   

As a practice dye, we use the bromophenol blue sample. The negative charge of the dye allows it to migrate off the gel and not interfere with the other dyes in the middle of the gel.

Prepare individual sample tubes for each student by adding 12 ul of the practice dye to a small microcentrifuge tube (0.5 or 0.65 ml size). We usually prepare one tube for each student.

Before the lab, label small microtubes (0.5 or 0.65 ml size) with numbers 1- 6. Prepare enough tubes for all the lab stations. In our labs, the students work in groups of four, and each group needs one of each numbered sample.

Before classes start, distribute the biological dye samples into the proper microtubes. Each tube gets 12 ul of the dye sample, enough for a single well. Make sure each dye is added to the correct set of numbered tubes. Store samples in refrigerator until needed.

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