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 micropipet (review Techniques Lab A ext) 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.

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

• 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.

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 polymerizes (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 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 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 2 µL of loading dye into the micropipet. (Remember: depress the plunger to the FIRST STOP before lowering the tip into the dye sample.)
  
  
• Steady the pipet over the well, using your second hand to support your pipetting hand or arm.

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!

9. Apply ONE drop of liquid soap to a piece of paper towel and spread evenly around the inside of the gel box LID. If previous periods already did this, do not add more soap. DO NOT GET THE SOAP IN THE BUFFER. 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. (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. Record the location of the dyes on your Activity Sheet, p. 2.
    
    
13. Turn off the power and disconnect the leads.
    
    
14. Save the buffer for reuse by the next class. Return gels to teacher for reuse after melting.
    
    
15. 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.

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