Gel Electrophoresis

Make the comb:

1. First, you'll need to make a comb to create wells in the gel that will eventually hold your samples. Rest a regular-sized craft stick on top of the small plastic box.
2. Cut the narrow craft sticks into several shorter lengths as follows: when taped to the side of a regular-sized craft stick, each segment should hang down so it sits just above the bottom of the small plastic box (approximately 1–1.5 inches (2–3 cm) in length depending on the depth of your box). Cut five of these "teeth" and tape them to one side of the regular-sized craft stick. Ensure that the teeth are evenly spaced, hang down to the same level, and all fit inside the box.
     
3. Tape the other regular-sized craft stick parallel to the first one, so that it creates a sandwich for the teeth, then lay the comb across the short side of the box. Check that the teeth hang evenly and don’t touch the bottom (click to enlarge diagram below).
   

Prepare your gel:

1. Make a 0.2% sodium bicarbonate buffer by dissolving 2 grams of baking soda in 1 liter of water. You will need approximately 100 milliliters per set up—half to make the gel and half to run your samples.
2. Make a 1% gel solution by adding 0.5 g of agar-agar powder to 50 mL of sodium bicarbonate buffer. You will need 40–50 mL of gel solution per set up. To dissolve the agar-agar powder, heat the gel solution in the microwave, stopping every so often to stir it. Watch the solution carefully, as it will quickly boil over when hot enough. When you see bubbles, stop the microwave and stir the solution until the agar-agar particles completely dissolve. The solution should be translucent and will solidify at room temperature.
3. Once the solution is cool enough to pour, add just enough to the box so that the comb teeth are submerged approximately 0.5 cm. Orient the box like a sheet of paper in portrait view, and position the comb across the narrow side of the box approximately 1.5 cm from the top. Thinner gels will yield better separations.

Set the electrodes in place:

1. Once the gel sets (approximately 5–10 minutes), you need to make space to place an electrode on either end. To make your electrodes, bend each piece of stainless steel wire so that it fits inside the box along its width and hooks over the side (image below). 
   
2. The actual gel only needs to be half the length of the box, so you can use a knife to cut away the bottom half of the gel that doesn’t include the comb. Being careful not to disturb the comb, also cut away a thin strip from the very top of the gel to make space for an electrode at the top of the box. Your gel should now be around 2.4 in (6 cm) long and 3 in (8 cm) wide—still the full width of your box. Place one electrode along the top end of the gel and one at the bottom end, using tape on the outside of the box if needed to secure them in place. These will be the positive and negative electrodes.
   

Set up your power source and prepare your samples:

1. Make a high-voltage power supply by connecting the five 9-volt batteries. Clip two batteries together by inserting the positive terminal of one into the negative terminal of another. Attach the remaining batteries one by one in this way until you have a five-battery pack.
2. Clip an electrical lead to each of the exposed terminals of the pack. You should now be able to use the battery pack to power something by attaching the other ends of the electrical leads.
3. Prepare five different samples by mixing 1–2 drops of food coloring with 1 mL glycerin and 1 mL water in a small tube. We use blue, red, green, yellow, and purple (made by mixing blue and red food coloring).

Once you have set up your gel, pour in just enough sodium bicarbonate buffer to cover the solidified gel. Make sure you fill up the spaces left from where you cut away the gel—the gel and stainless steel wires should be completely submerged.

Gently remove the comb by pulling straight up without tearing the gel. The wells left by the comb should fill with buffer.

Use the needle-tip pipette to transfer approximately 10 microliters (µL) of each sample to an empty well. (The volume of the thin tip of the pipette is about 10 µL.) Submerge the tip in the buffer directly above the well and gently squeeze the sample into the well. It should fall into the well since it is denser than the surrounding buffer. Be sure to use a new pipette for each sample to prevent contamination between samples. If you have a limited supply of pipettes, thoroughly rinse out the tip in a large beaker of water before reusing.

Once all the samples are loaded, connect the leads from the power supply to the stainless-steel wire electrodes attached to the box. Connect the negative terminal to the electrode at the top of the gel (near the wells) and the positive terminal to the electrode at the bottom of the gel. You should see bubbles forming along the electrodes when a complete circuit is made.

Leave the power connected for 15–20 minutes and observe what happens to each sample. 

Gel electrophoresis is one of the most important tools used in molecular biology and genetic engineering. By conducting an electric current through an electrolyte buffer—like the sodium bicarbonate buffer that you used—charged molecules will migrate towards the terminal with the opposite charge. When suspended within a polymer matrix—such as an agar-agar gel—the molecules will move at different rates based on their size.

In this experiment, negatively charged dye molecules are loaded into the gel. When a current is passed through the gel, the molecules migrate towards the positive terminal, with smaller molecules moving faster than larger ones. This separates the different color molecules.

Look at the ingredient list of the food coloring you used to see what molecules are inside. Different brands can use different dyes. In the pictures shown here, we used Smart & Final brand dyes, which contain Blue #1, Red #40, and Yellow #5. Using gel electrophoresis, we discovered that McCormick-brand blue food coloring is made of a blue dye plus a small amount of red. McCormick red dye is actually made of two different red-colored molecules. Test different brands to discover the true composition of each color.

In molecular biology, this technique is used to separate biological compounds, such as DNA or proteins, based on their size. Like the colored dyes, DNA and proteins are negatively charged, so they will migrate towards the positive electrode at different speeds depending on their size.

Scientists can use special enzymes to cut a large strand of DNA into many smaller pieces. The size of the pieces will depend on the specific base sequence of the large DNA strand. This technique is used in DNA fingerprinting to identify people since each individual’s gene sequence will result in a unique “fingerprint” of DNA bands. 

You can recycle the pieces of gel that you cut out before adding your samples. Simply reheat them in the microwave, and they will turn back into liquid.