Dye Electrophoresis

Advanced Preparation

Agarose: Making up 2% agarose (notice this is agarose and NOT agar) can be done at any time, even weeks in advance. The agarose must be made using FRESH 1XTBE buffer NOT water. It does not have to be autoclaved only melted (clear not clouded). It can be stored in closed bottles at room temperature and melted by using a microwave oven or hot water bath. Be sure to loosen the bottle's cap before heating.Using the microwave is much simpler and quicker.
Gels: The volume of 1XTBE buffer needed to make 20 gels is approximately 800 ml. To pour the gels, you will need this volume of agarose times the number of classes doing this activity. You should pour the gels a few days in advance of the lab date. When taping the ends of a gel tray, be sure that the masking tape is pressed firmly along the edges of the tray. This will prevent leaks. Pouring the agarose should be done at approximately 60-65ûC (Caution Hot). This temperature can be approximated by feel. If you can hold both hands around the bottle and not get a burning sensation, the agarose is at the correct temperature. However, for added protection, use hot gloves when pouring the gels. You should have as many gel trays prepared as you have agarose . Each gel requires 35-40 ml of agarose. Line the gel trays along the edge of a lab table and pour the agarose into one side. Pour until the agarose comes within 3/4 of an inch of the empty end. Stop pouring and allow the agarose to flow to the end of the tray. This exercise requires that the gels have at least 6 wells and that the Gel Comb be placed in the Center of the tray. The gels can be stored inside zip lock bags at room temperature or in the refrigerator. Pour one or two extra gels.Students who make major mistakes can be handed a new one to complete the exercise. Since this lab is designed to get results within 30-40 minutes, it is essential that the gels be poured ahead of time.
Buffer: In addition to the 1XTBE buffer needed for the gels, you will need to make up approximately 500 ml for each electrophoresis chamber. 8 L of 1XTBE buffer should be enough for 16 chambers. You can reuse the 1XTBE buffer from the chambers so long as it is not badly contaminated by any student team. If contaminated, discard and replace with fresh 1XTBE buffer. If you have highly motivated or advanced students, gel pouring and buffer dilution might be a special project for them to do as an after school exercise.
Tip boxes: Be sure that all the micropipet tip boxes are filled. These tips do not have to be kept sterile so it's OK to handle them without gloves. This task is another one that can be done by your advanced students after school or student service person.
Dye kits: The dye kits need to be ordered several weeks in advance. They can be purchased from Ward's Natural Science Establishment catalog # 36W5254. These dyes are very inexpensive and can be used for several labs. The kit contains the following: Bromphenol Blue, Janus Green, Orange G, Safranin O, Xylene Cyanol and a tube of some mixture of these 5 dyes. Each tube contains approximately 1 ml of dye. You will have to transfer 100 µl of each dye to microcentrifuge tubes (1.5 ml) and make a set for each team. Place each set of dyes in the microcentrifuge tube rack and place at each lab station. Since the exercise requires 10 µl of each dye per gel, one set is sufficient to supply a team of 2 students for up to seven periods.
Preparation time: Estimated time to make and melt agarose is 1 hour. Estimated time to pour gels (16 for a class of 32) is 15-30 minutes. Estimated time to label microcentrifuge tubes and transfer dyes is 30-45 minutes. Estimated time to make up l0 L of 1XTBE buffer is 30 minutes. Total estimated prep time 11/2-2 hours.

Introduction

This exercise allows students to learn the technique of molecular separation by use of gel electrophoresis. They will learn the principals that allow separation by this method and how to use another important tool of the molecular geneticist.

Student Objectives

The student will use the electrophoresis equipment.
The student will demonstrate how charge and size affect a molecule's movement through a gel matrix.
The student will use the principles of electrophoresis to seperate different molecules from a mixture.

Class Time Needed

Two 50-55 minute class period is required to run the lab.

On the first day students should load the gel and collect data.
Day two is for data analysis.

Materials

Electrophoresis chamber
Power supply
2% Agarose gel (centered wells)
Micropipet (1-20 µl)
Dye set (6 tubes)
Pipet tip box
Large funnel
Waste container
1XTBE buffer solution
Microcentrifuge tube rack
1/2 inch masking tape
Gel tray and comb

Recipes for Consumables

1XTBE buffer is made by diluting 100 ml of 10XTBE stock solution in 900 ml of distilled water.

2% agarose is made by melting 2 g of agarose powder melted in 100 ml of 1XTBE solution.

Procedure

Since this is the first time students will be placing the gel and 1XTBE buffer into the electrophoresis chamber, you should model how to do this step and then check every team. You must be sure that the wells are full of buffer, but the level of the buffer is just high enough to barely cover the gel. Be sure the students press down on the edges of the gel tray until it sits down on the platform in the electrophoresis chamber. This will prevent floating trays and gels.

Loading the gel should not be a problem; however, you may wish to quickly review the micropipet and its use. Pay special attention to which stop for drawing up a liquid and which stop for expelling it are probably the most important items to review. Lane 1 is usually defined as the outermost well of the gel on the black electrode side of the electrophoresis chamber. Once this well is loaded, the students can then load the wells in sequence according to the procedure.

Closing the electrophoresis chamber and connecting it to the power supply deserve special attention. Be sure that the lid goes on black lead wire to black electrode and red lead wire to red electrode. Also, be sure that the electrophoresis chamber is positioned where you want it on the table. Once the chamber has been connected to the power supply and the power has been turned on, the student should NOT touch or handle the chamber. Check the lead wires from the lid of the chamber to be sure that they have been plugged into the correct receptacles of the power supply. Black to black and red to red and that the wires are plugged into receptacles that are next to each other. Once you have checked all of this at a lab station, you can turn on the power supply and start the experiment running. Set the power supply to a constant 100 volts and use this as the running voltage.

Once the dyes have sufficiently migrated through the gel, usually 20 minutes, turn off the power supply. Be sure that the students remove the black and red lead wires of the electrophoresis chamber lid from the power supply BEFORE they remove the lid from the chamber.

Remove the gel from the gel tray and place it on a blank, white sheet of paper in order to see it more easily. Recording the data from the gel MUST be done on the same day as they were run. The dyes diffuse so rapidly throughout the gel that you can not leave it for the next day.

Even though having the students follow the clean up procedure every period makes more work for you, it is important that each team in every period learn how much 1XTBE buffer to add to the electrophoresis chamber and how to properly clean their work station. Be sure that the students DO NOT attempt to wipe dry the inside of the electrophoresis chamber. Wiping the inside of the chamber may result in snagging and breaking the platinum wire electrode that runs the width of the chamber at each end.

Disposal

Agarose gels can be placed directly in the garbage. It is probably best to put all the gels in a zip lock bag before placing them in the garbage.
Tubes containing the dyes can be saved for future use.
Be sure to save the 1XTBE buffer from the electrophoresis chambers for future use.