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From the DNA Kit Program Manual

Ever wonder how the banding patterns in DNA paternity or forensic cases are formed? Using a process known as gel electrophoresis, DNA fragments can be separated based on their size and charge.

Gel Electrophoresis At a Glance

Goal: To give visitors an opportunity to understand the process used in sequencing a genome
Level: Introductory to advanced
Age: Middle school visitors and above

Molecules can be separated by size and charge using a method known as agarose gel electrophoresis. It is one of the most common methods used by researchers to isolate and identify fragments of DNA. Agarose forms a matrix that acts as a sieve through which smaller molecules can migrate faster than larger ones. Different matrices can be used to separate DNA molecules that differ by only a single base pair or are chromosomal in size.

To prepare an agarose gel, agarose is heated in a buffer solution and poured into a gel-casting chamber fitted with combs creating a series of sample wells. As the gel cools to room temperature, it becomes a semi-solid matrix (much like Jello) with microscopic holes. Samples are loaded into the wells and an electrical field is applied across the gel. This causes the molecules to migrate through the gel toward one of the electrodes (positive or negative) depending upon their charge. Most proteins and DNA are negatively charged and move toward the positive electrode. Separation of molecules of different sizes can take only a few minutes or a few hours depending upon the agarose concentration and the voltage applied to the gel. Gels containing DNA fragments are typically stained with a DNA sensitive dye such as ethidium bromide or bromophenol blue. The gels can then be documented by photography when illuminated under UV or white light.

The following exercise provides a basic introduction into the theory and practice of gel electrophoresis using samples prepared from food coloring. Idealized results should demonstrate that all complex colors (purple, green, orange) are made up of the primary colors red, blue and yellow. These colors each have a distinctive molecular size that can be separated using electrophoresis. Separation of the colors occurs very quickly (5-10 minutes) and is appropriate for children of all ages. Younger participants may need assistance in handling the micropipet and loading samples but everyone loves the tip ejector!

Equipment and Supplies

• 50X TAE (Tris-acetate-EDTA).buffer (recipe follows)
• Electrophoresis chamber and casting tray, combs
• Micropipettor and tips
• Power supply
• Prepared food coloring samples (recipe follows)
• Beaker or waste container for used tips
• Clean container of water if tips are to be rinsed and reused Agarose

Things you will need that are not supplied as part of this kit

• Distilled water
• Plastic cups or disposal containers for pipet tips
• 1 liter container for gel buffer
• Microwave oven or heating plate
• 250 ml flask

Activity Set-up

1. Dilute 50X TAE with distilled water. Using a graduated cylinder, measure out 20 ml of 50X TAE buffer. Dilute to 1 liter with distilled water. Store tightly capped at room temperature.
2. Remove contents of electrophoresis box and place on the table.
3. Unpack the power supply and plug it into an outlet.
4. Prepare agarose gel.
5. Fill the electrophoresis box with 1X TAE buffer to a level that barely covers the entire surface of the gel.

Preparation of the Agarose Gel

Makes 150 ml. Gels can be made ahead of time and stored under damp towels in a sealed container at 4-C.

1. Weigh out 1.2 grams agarose (for a 0.8% gel). This is enough to fill six individual casting trays. Place in a 250 ml flask.
2. Add 150 ml of 1X TAE
3. Melt the agarose using one of the following two methods:
   1. Heat in a microwave on high for one or two minutes, swirl the solution at one minute.
   2. Cover the flask with aluminum foil and heat in a boiling water bath until agarose is melted.
4. Seal the ends of the gel-casting tray with rubber dams, and insert the well-forming comb. Place the gel-casting tray out of the way on the lab bench so that the agarose can set undisturbed.
   
   Figure 4. Casting tray with comb and rubber dams in place.
   
5. Allow the agarose to cool slightly before pouring into the casting tray. Hot agarose can cause the tray to crack.
6. Carefully pour enough agarose solution into the casting tray to fill it to a depth of about one-third the height of the comb teeth. While the gel is still liquid, use a pipet tip to move large bubbles or solid debris to the sides or end of the tray.
7. The gel will become cloudy as it solidifies (in about 10 minutes). Be careful not to move or jar the casting tray while the agarose is solidifying. Touch a corner of the agarose away from the comb to test whether the gel has solidified.
8. When the agarose has set, unseal the ends of the casting tray. Slide a small metal spatula or the end of a pipet tip between the agarose and the rubber dam to release the seal formed between the dam and the gel.
9. Gently remove the comb, pulling it straight up and out of the set agarose. Do not rock or wiggle the comb.
10. Store the agarose gels in a tightly sealed, refrigerated, humid environment until ready to use. 
11. Gels can be loaded either in the electrophoresis chamber with buffer around them or on the bench top. If you load the gels on the bench top, be sure to fill the wells with buffer beforehand.
12. Place the casting trays on the platform of the electrophoresis box, so that the wells are at the negative (black) electrode end matching the notches in the chamber.

Figure 5. Proper alignment of the casting tray in the electrophoresis chamber

Things to think about when using this activity

• Each casting tray has six sample wells. With the six casting trays this provides opportunities for 36 visitors to run samples at the same time. Once the colored samples reach 2/3 of the way down the gel, another set can be loaded into the wells. The previous samples will run off the bottom of the gel into the buffer solution. This allows many samples to be running continuously.
  
  
• You can choose to load samples into the agarose gels either "wet" or "dry". "Wet" is when the casting trays are placed in the electrophoresis chamber and buffer is added just up to and filling the wells. "Dry" is either on the bench top or in the electrophoresis chamber without buffer. If you choose to load your sample using the dry method, be sure to fill the wells with buffer prior to loading your samples. Be very careful when placing the casting trays in the electrophoresis buffer or filling the chamber up with buffer or you will lose your samples.
  
  
• When loading samples into the wells, the micropipet tip should be positioned in the center of the well just below the surface of the buffer. You do not want visitors poking the end of the tip into the agarose at the bottom of the well. The gels are fragile and pipet tips can dig into the bottom of the well, poking all the way through. It is sometimes helpful if the volunteer or staff personnel help to position the tip in the well by holding onto the shaft of the micropipet. Encourage your visitors to brace their arm on the table top and use their other hand to steady the tip.
  
  
• Once a gel is fully loaded and ready to run, it should be placed in the electrophoresis chamber and gently surrounded by buffer. The buffer level should come up just to the top of the wells. Be careful when placing the trays in the chamber that you don't plop them down and swish away all of the colored samples.
  
  
• It typically takes about 5-10 minutes before the colors begin to separate. You should see bubbles forming on the metal leads once the power is connected. Separation is usually complete in 20-60 minutes.
  
  
• This activity does involve the use of electric current at high voltage. Even though there is an interlock remember to caution your visitors when using the equipment.

Procedure

The samples used in this exercise consist of food coloring (a few drops) in a buffer solution with a few drops of glycerin to help the sample sink to the bottom. You can make up your own color combinations, or use the prepared samples.

1. Firmly set a pipet tip onto the end of the 25 -l micropipet.
2. Holding the sample tube in one hand, flip up the top of the tube with your thumb.
3. Push down on the plunger of the micropipettor to the first stop and hold it in place while placing the tip into the sample tube. Don't push the tip too far down into the sample solution or the food coloring will adhere to the outside of the tip and leak onto the gel.
4. Release the plunger and remove the micropipettor tip from the sample tube.
5. With your thumb, snap the cap back onto the tube and place the sample tube back into the rack.
6. Load your sample into a clean well slot.
   1. Steady the pipet over the well, using two hands.
   2. If there is air in the end of the tip, carefully depress the plunger to push the sample to the end of the tip. (An air bubble ejected into the well can form a "cap" over the well, causing the DNA/loading dye solution to flow into the buffer around the edges of the well.)
   3. Center the pipet tip over the well, dip the tip in only enough to pierce the buffer surface, and then gently depress the pipet plunger to slowly expel the sample. Take care not to poke the end of the pipet tip into the bottom of the well. The tips are sharp and can poke a hole in the fragile gel.
7. Eject the used tip into the proper waste container or rinse the tip in a cup of clean water. Eject the tip back into the box or a holding container.
8. When all the samples have been loaded into the gel, gently place the tray into the electrophoresis chamber filled with buffer.
9. Place the lid on the electrophoresis chamber, and connect the electrical plugs to the power supply. You should begin to see separation of the colored molecules within 5-8 minutes.
10. Allow the gel to run until all the colors are separated, about 15-20 minutes.
11. Unplug the power supply, disconnect the plugs and remove the cover from the chamber.
12. The gel can be saved for another day or disposed of in regular trash. The TAE buffer can be saved for additional electrophoresis activities.

Activity clean up

1. Unplug power supply. Remove leads from electrophoresis chamber.
2. Carefully pour running buffer into storage container. Buffer can be reused many times before it diminishes in effectiveness. Dispose of used buffer down the drain.
3. Rinse chamber with clean water.
4. Clean out agarose from casting trays. Agarose can be disposed in regular trash. Rinse casting trays with clean water.
5. Dispose of used tips in trash or rinse for reuse.

Questions that may come up about this activity

What does this exercise tell you about the properties of the red, blue and yellow molecules in relation to their size and charge?

All of the molecules are negatively charged, since they migrate toward the positive (red) electrode. From their migration patterns, the yellow molecule travels the furthest, followed by the red and the blue. Therefore, the yellow molecule must be the smallest, followed by red and the blue molecule is the largest of the three.

What would happen if the chamber contained water instead of a buffer solution?

If the chamber contained water, there would be very few ions present to establish an electric field and thus no current produced. The food coloring molecules would not move through the gel and would stay in one place.

How would a higher agarose concentration change the migration of molecules through the gel?

Higher concentrations of agarose (1.0-1.5%) are used to separate smaller fragments of DNA because the pore sizes in the gel matrix are smaller. A higher concentration of agarose would cause the smaller bands to move more slowly through the gel.

Measure the distance each color migrated through the gel. Can you make some estimates on the size of each of these molecules based on the distance traveled?

The size of each molecule is roughly proportional to the inverse log10 of the distance traveled.

Recipes for Buffers and Solutions

50X TAE can be ordered from many different suppliers. The buffer provided with this kit was provided by Edvotek (catalog # 607-1L). If you would like to make up your own buffer solutions, the recipe is given below.
50X TAE (Tris/acetate/EDTA) Electrophoresis buffer
Makes 1 liter. Store at room temperature.

1. Measure ~900ml distilled H2O.
2. Add 242g Tris base (m.w. 121.1, Sigma catalog #25,285-9)
3. Add 57.1ml Glacial Acetic Acid.
4. Add 18.6 g EDTA (ethylene diamine tetraacetic acid, disodium salt, m.w. 372.24, Sigma catalog #E2,628-2)
5. Adjust volume to 1L with additional distilled H2O.

Food coloring samples

In a 1.5 ml microfuge tube place one or two drops of food coloring, mixing primary colors to give complex colors. Dilute to 0.5 ml with 1X TBE. Add an equal volume of glycerol (0.5 ml) and mix well.

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Last Reviewed: October 25, 2007