Gel electrophoresis is an important molecular biology tool. DNA sequencing, fingerprinting (or "profiling"), and genetic engineering are based upon it. Gel electrophoresis separates DNA fragments by their size or molecular weight. The agarose gel acts like a sieve, separating different sized fragments while the electric current provides the driving force. DNA, a negatively charge molecule, is attracted to the positive electrode; the voltage determines how fast the DNA will travel through the gel. Larger molecules or DNA fragments become entangled in the gel and travel more slowly, while smaller ones pass through more easily and travel farther down the gel. Similar-sized DNA fragments travel at the same rate and form a tight bunch called a "band". The DNA in the gel must be stained in order to see the bands.

1. Remove the comb from the wells by pulling straight up on the comb. Be careful not to tear the wells as you remove the comb. Remove the tape from both ends of the gel tray.
   
   
2. Place the gel tray in the gel box with the wells closest to the negative (black) electrode.
   
   
3. Add enough 1X TBE buffer to fill the buffer tank and submerge the gel about 1/4 inch.
   
   
4. On a piece of wax paper, mix 5 uL of gel loading dye with 10 uL of your DNA sample. Mix the solution by pipetting the dye up and down into the 10 uL DNA sample. The gel loading dye contains glycerol which will make your DNA more dense so that it will sink into the wells. It also contains dye molecules which are smaller and travel faster through the gel than the DNA molecules. The dye molecules provide a visual tracking method so you know how far the DNA has traveled through the gel.
   
   
5. Rinse the pipette or capillary tube with clean water by gently pipetting up and down two or three times.
   
   
6. Make a sketch of the gel; include all of the wells and the positive (red) and negative (black) electrodes. Indicate under each well the name of the sample to be loaded.
   
   
7. Pipette 10 uL of the first sample, usually a reference sample such as a 1 kb ladder, into the first well. Keep the tip of the pipettor ABOVE the well. The DNA will sink into the well because it has been mixed with loading dye. If you puncture the bottom of the well your DNA run out the bottom of the gel into the buffer tank.
   • Molecular biologists often use a size standard called a 1 kb DNA ladder. The DNA ladder produces several different sized fragments or bands and can be used to estimate the size of an unknown DNA fragment.
     
     
8. Rinse the pipette or capillary tube in the buffer tank by gently pipetting up and down two or three times.
   
   
9. You are now ready to load the next sample into the next well. Repeat steps #7-8 until all of the samples and controls have been loaded into the gel. Your teacher will tell you how many lanes to save for concentration standards and which ones to load. Remember to record on your sketch the order the samples were loaded (including standards!)
   • A concentration standard is used to estimate how much DNA is present in a sample.
     
     
10. Close the top of the box. Plug the leads into the gel box. The black lead is the negative lead and should be plugged in closest to the wells. The red lead is the positive lead and should be plugged in furthest from the wells.
    • Remember: Red ahead.
      
      
11. Plug the other end of the leads into the power source and turn it on. Run the gel at between 80-120 volts until the loading dye has traveled 1/2 - 3/4 of the way down the gel (about 30-45 minutes).
    
    
12. Turn off the power supply. Unplug the leads and the power supply before opening the gel box.
    
    
13. The gels may be wrapped in Syran Wrap and stored overnight until they can be stained.
    
    
    
    

Science Education Connection Department of Biochemistry The University of Arizona Tuesday, January 14, 1997 warder@u.arizona.edu http://biology.arizona.edu/sciconn/lessons/vuturo/ All contents copyright © 1997. All rights reserved.