Techniques Lab C-2: Casting and Loading an Agarose Gel
Separation of Assorted Charged Dyes
In Techniques Lab B, you investigated the components of electrophoresis.
You learned that 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.
The comb is removed, creating empty wells. Then, a micropipet (review Techniques Lab A exrt)
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. You will load these two dyes on your gel. In addition, your instructor will put out a variety of dyes for you to test in the gel system. Your task is to predict whether the dyes are positively or negatively charged, then observe and analyze the results.
To practice the steps required in the casting an agarose gel and loading tracking dyes.
Materials per team
||buffer [1X TAE or 1X TBE]
||glass beaker, 50 mL, for agarose
|gel box with gel tray
||assorted dyes in glycerol
||beaker, 500 mL, for buffer
|P-20 micropipet & tips
||container for waste (tips)
|Paper towels/Kim wipes
|agarose, [0.8%], melted and kept
hot in a 65°C bath or incubator
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.
Procedure: Casting a Gel
- 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 middle slots of the empty tray.
- Place the prepared casting tray on a paper towel.
- Obtain a beaker with 25-30 mL of liquid agarose, which has been kept at 65-70°C
in a water bath. Pour the agarose evenly into the casting tray.
- DO NOT JAR or MOVE the casting tray as the gel solidifies. This ensures a smooth,
even gel. As the agarose polymerizes (about 10 minutes), it changes from clear to slightly opaque.
- While you are waiting, fill the electrophoresis box with about 300 mL of 1X TAE (or TBE) electrophoresis buffer. (This may have been done by a previous class.) ORIENT THE GEL BOX SO THAT THE WIRE LEADS ARE FACING YOU.
- 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 in the center. The level of the buffer should be only a few mm above the surface of the gel.
- Carefully 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. Be sure there is no "dimpling" of the
buffer above the wells. Add more buffer if needed.
Loading the Gel
- 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 toward 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):
Electrophoresis of the Dyes
- Draw 2 µL of dye into the micropipet. (Remeber: depress plunger to the FIRST STOP before lowering the tip into the dye sample.)
- Steady the pipet over the well, using two hands.
- Lower the tip of the pipet 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 pipet 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 pipet plunger depressed to the SECOND STOP until the pipet tip is out of the gel box or you'll draw your sample back into the tip!
Upon completion of this lab
- Apply a drop of liquid soap to the inside of the gel box LID and spread evenly. 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.
- Set the power supply to approximately 100 V, and turn it ON. (As a check: about 40 milliamps of current should be registering on the display if one gel is running; two gels will run at about 80 milliamps with 1X TAE buffer.) This confirms that current is flowing through the gel.
- Shortly after the current is applied, you should see the dyes moving through the gel.
- Electrophorese for about 10 minutes. Record the location of the dyes on your
- Turn off the power and disconnect the leads.
- Save the buffer for reuse by the next class. Return gels to teacher for reuse after melting.
- Return gels to teacher for reuse after melting or let dry on white blotter paper or paper toweling to save a "color blot" of your gel.
- Record your observations on your Activity Sheet.
- 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