1. Measure the gel plate you intend to use and determine the volume of solution needed to prepare a 3 mm thick gel. Tape the edges of the plate to form a mold to hold the gel solution. (For small gel plates, the hot agarose will stay on the plate by surface tension so taping may not be necessary). Position a comb above the gel plate, leaving a 0.5-1 mm space between bottom of teeth and gel plate so that the wells actually have an agarose bottom.

2. In a flask, add powdered agarose to the appropriate volume of 1X TBE. Melt the agarose in a boiling water bath or a microwave oven. If using a microwave, watch the solution carefully to prevent boil-over. Shake the flask occasionally to ensure mixing. Use a flask that contains at least twice the volume of the agarose solution you'll have in it. For example, I use a 500 ml flask when preparing 150 ml of agarose.

3. When the agarose is melted, cool the solution to 60oC. This is most easily done by placing it in a 50o to 60o C water bath. Once the gel has cooled ethidium bromide can be added to 1 mg/ml. Pour the gel.

4. After the gel has set (1/2 to 1 hr), remove the comb and place the plate in the gel box. Take care when removing the comb. When you pull the comb out, a vacuum is created, and if you are not careful, you can pull the bottom of the wells up. This can be a disaster as you samples will leak out of the bottom of the wells. Orient the gel so that the wells are at the negative electrode. Remember, DNA and RNA run toward the positive poles due to all those negatively charged phosphates.

5. Barely cover the gel with 1X TBE. If the samples have not had a loading buffer mix added to them, add 1/10 volume of loading dye to each sample and mix thoroughly. Load the DNA samples and begin the run. Recipes for two popular loading buffers are shown below. Remember that the speed with which molecules move in an electric field is a direct consequence of the voltage. Current is related to the voltage, and the resistance of the gel. It is directly proportional to the heat generated during the gel run. It is common to run DNA gels any where from 50 to 150 volts. Just pay attention to how hot the gel gets.

6. After the dye has run the desired distance, disconnect the power supply from the gel and remove the gel. Carefully slide it into a tray containing a solution of 1 mg/ml ethidium bromide (EtBr). Alternatively, EtBr can be put into the gel so the DNA is stained during the run. Ethidium bromide works by intercalating between the bases in DNA. This property also makes it an effective mutagen. Be careful with it. Never mouth pipette and always wear gloves when handling it. Twenty to thirty minutes in ethidium bromide followed by rinsing with distilled water and a brief incubation in distilled water will produce the best looking gels. Alternatively, ethidium can be put into the molten agarose just prior to pouring the gel. In this case, the gel can be viewed during the electrophoresis.