Electrophoresis (a term which literally means "to carry with electricity") is a technique for separating and analyzing mixtures of charged molecules. Clearly this separation wouldn't work very well if the molecules were just sprinkled on the surface of the gel box fluid! Instead, the mixture to be separated is "loaded" into slots or "wells" of a slab of jelly-like material called agarose. Agarose is a very pure form of agar, which is actually made from a kind of seaweed. To prepare or "cast" an agarose gel, agarose powder is mixed with buffer, heated, and poured into a casting or gel tray containing a comb. When the gel has cooled and solidified, the entire casting tray is lowered into the gel box and covered with buffer which allows the electricity to flow and prevents changes in pH. The comb is removed, creating empty wells. Then, a is used to place a small amount -- usually just a few microliters -- of the mixture to be separated into each well.

In order to track where the "invisible" DNA runs on a gel, we add two dyes to the DNA sample. One dye runs slightly faster and farther than DNA; the second dye runs slower and not as far as the DNA.

To practice the steps required in the casting and loading of an agarose gel. Observe the migration of DNA samples, standards, and dyes commonly used to track DNA in agarose gels.

Materials per team (Note: all items can be found in Ward’s Scientific Catalog)

Power supply (either variable power or 5-10 nine volt batteries joined in tandem

buffer [1X TAE or 1X TBE]

glass beaker, 50 ml for agarose

gel box with gel tray

loading dye

beaker, 500 ml, for buffer

P-20 micropipet & tips

container for waste (tips)

Paper towels/Kim wipes

agarose, [0. 8%], melted & kept hot in a 65 degree C bath or incubator

Quickview stain or Ethidium Bromide (see warning below for EtBr)

If two teams are connecting their gel boxes to one power supply, be sure to communicate with each other whenever the power supply is turned ON or OFF. The power supply must be OFF every time anyone needs to touch or open a gel box.

1. Loosen the screws at the ends of a casting tray, if necessary, to raise the "gates" at each end; then, tighten the screws (not too tight) until there is enough tension to hold the gates in place. Insert a comb in the end slots of the empty tray. Comb must be at the bottom when the longer side of the gel tray is held in your right hand. See diagram below.

2. Place the prepared casting tray on a paper towel.

3. Obtain a beaker with 25-30 ml of liquid agarose, which has been kept at 65-70 degrees C in a water bath. Pour the agarose evenly into the casting tray. DO NOT POUR THE GEL IF AGAROSE IS ABOVE 70 degrees C. Clean and dry the beakers, making sure that none of the agarose is left.

4. DO NOT JAR or MOVE the casting tray as the gel solidifies. This ensures a smooth, even gel. As the agarose hardens (about 10 minutes), it changes from clear to slightly opaque.

5. While you are waiting, fill the electrophoresis box with about ~300 ml of 1X TAE or TBE (whichever was used to prepare the agarose gel) electrophoresis buffer. (This may have been done by a previous class. ) ORIENT THE GEL BOX SO THAT THE WIRE LEADS ARE FACING YOU.

6. When the gel has solidified, lower and secure the "gates" at both ends of the casting tray. USING THE HIGHER SIDE OF THE CASTING TRAY AS A HANDLE, TIP THE TRAY SLIGHTLY AND INSERT THE LOWER SIDE OF THE TRAY TO THE REAR OF THE BOX. Submerge the tray onto its platform in the gel box. The comb should be located at the cathode end (black lead; (-) end). The level of the buffer should be only a few mm above the surface of the gel.

7. Carefully and slowly remove the comb from the gel (pull it straight out). You'll notice that this left behind six little empty "slots" or wells in the gel. To check to see if there is enough buffer, look to see that there is no "dimpling" of the buffer above the wells. Add more buffer if needed.

8. By convention, DNA gels are read from left to right, with the wells located at the top of the gel. With your gel lined up in its box with the wells to your left, the contents of Tube "1" should be loaded in the well closest to you. Thus, when the gel is turned so that the wells are at the top, "1" will be in the left-hand corner.

Use a P-20 micropipet to practice the technique of loading a well (there are several wells in one gel, so every member of your team can practice this):

Draw appropriate amount of DNA or ladder (measure in µL) into the micropipet. (Remember: depress the plunger to the FIRST STOP before lowering the tip into the dye sample. )

Steady the pipette over the well, using your second hand to support your pipetting hand or arm.

Lower the tip of the pipette under the surface of the buffer directly over the well -- but do not lower the tip into the well itself, or you risk puncturing the bottom of the gel.

Gently depress the pipette plunger to slowly expel the loading dye into the well. If the tip of the micropipet is centered over the well, the dye will sink to the bottom of the well.

REMEMBER - keep the pipette plunger depressed to the SECOND STOP until the pipette tip is out of the gel box or you'll draw your sample back into the tip!

9. Close the top of the gel box and connect electrical leads anode to anode (red to red) and cathode to cathode (black to black). Both electrodes should be connected to one power supply channel.

10. Set the power supply to approximately 100 V, and turn it ON (or connect to 9V batteries). (As a check to see that electricity is flowing, look for bubbles at the wire at either end of the gel box. )

11. Shortly after the voltage is turned on, you should see the dyes slowly moving through the gel toward the positive side of the gel box.

12. Electrophorese for about 10 minutes, or until lowest dye migrates a little over half way through the gel.

13. Turn off the power and disconnect the leads.

14. Save the buffer for reuse by the next class.

15. Stain gel using Ward’s Quickview stain to view DNA or ladder (Note: Ethidium Bromide may be used and viewed under UV light, but caution should be taken. EtBr is an interterculator [binds within the twisted double helix of DNA] and suspected carcinogen)

Dispose of designated materials in the appropriate places.

Leave equipment as you found it.

Check that your work station is in order.

Wash your hands.

One FULL period. Explain lab procedures the day before you do the lab.

Mix one-two batches of 0. 8% (or appropriate concentration) agarose in 1X TAE buffer (not water!).

For example, 400 ml of 0. 8 % agarose would be enough for 15-20 teams, each using 20-25 ml. To make this, mix 3. 2 g of agarose power in 400 ml of 1X TAE buffer. 0.8% agarose resolves DNA fragments ranging in size from 0.6 to 35 kb, 1.0% for fragments 0.5 to 20 kb, and 2.0% for fragments 0.1 to 5 kb. The higher the percentage, the stiffer and more easy it is to handle the gel.

Check that loading dye has been aliquoted. 100 µL of loading dye per team will be enough. The loading dye contains 0. 25% xylene cyanole (XC) and 0. 25% bromphenol blue (BPB) in 50% glycerol, 10 mM Tris, 1 mM EDTA, pH 8. XC is a negatively charged dye that migrates slower than most DNA; BPB is a negatively charged dye that migrates faster than the DNA sizes we use. Glycerol is used to increase the density of the loaded sample so that the sample sinks down into the wells.

Carboys with 1X TAE should be set out in the classroom. To expedite dispensing of buffer, provide several beakers near the carboys.

Only the first class of the day needs to fill gel boxes. 1 X TAE will "last" for an entire day; it should NOT be discarded at the end of each period! Gently slosh the boxes to mix the ions.

Nothing needs to be sterile for this lab.

Show how to raise or lower, and secure, the "gates" on the ends of the casting trays. Unless you tell them not to, students may remove the screws entirely. They need only be loosened. Students might also over-tighten the screws. Screws should be adjusted so that the gates are snug with the edges of the tray, yet the gates can be moved up and down with reasonable effort. Once the tension on the gates has been properly adjusted, it is not usually necessary to loosen and re-tighten the screws with each use.

Review how to properly slide off gel box lid. DO NOT PULL ON WIRES!!!

Review how to use pipette for tiny volumes of viscous solutions.

Demonstrate how to load a well without "puncturing" the bottom of the gel with the pipette tip.

Careful -- loading dye tubes "splatter" as they are opened. The dye stains hands and clothes.

KEEP TRACK OF COMBS! They cost $10. 00 each!!! (You might want to color the edges with red or green marker to make the combs more visible, especially on white paper towels!)

It is worth mentioning that there are many types of electrophoresis systems, but they all function on the same principal discussed in this protocol.

Dissolve (or re-liquefy) agarose by microwaving. It takes only 5-6 min. on MEDIUM to liquefy a 500-ml bottle of agarose. THE LID MUST BE LOOSE. After heating, do not swirl too vigorously, or superheated agarose can bubble out of the bottle. Microwaving without LOOSELY covering causes evaporation and subsequent higher concentration gels!

HOLD melted agarose in a 65 degrees C water bath for use during the period. Students dispense what they need (20-25 ml), insuring that the agarose will not be so hot that it warps the casting trays as it is poured.

20-25 ml is a trade-off between gel running time (thinner runs faster) and gel durability (thicker gels are easier to handle without tearing).

A common error: wells of gel at wrong end of gel box. Loading dye will migrate toward the positive end of the box (anode), so the wells of gel should be at the cathode (negative; black) end of the gel box.

You can make a mixture of 4 dyes: bromphenol blue, xylene cyanole, phenol red and Orange G. By varying the combinations, you could have students determine which samples have the same dyes in them.

Another extension or modification of this activity is to put the wells in the center of the gel and have students load various dyes, some with positive charge, some with negative charge, and observe the dye migration.

At the end of the day, after all classes have completed the lab, the 1X TAE and the used agarose can be discarded. Agarose can be disposed of in everyday trash, and buffer can be disposed of in the sink (should be washed down with water to prevent salt build-up in sinks).