DNA gels are made of agarose, a highly purified agar, which is heated and dissolved in a buffer solution. The agarose molecules form a matrix with pores between them. The more concentrated the agarose, the smaller the pores. We will be using a 0.8% agarose gel (there are 0.8 grams of agarose per 100 mL of buffer) because we are looking at large DNA fragments thousands of base pairs in length. A 2% agarose gel separates DNA fragments which differ in length by as few as 50-100 base pairs.

Before beginning, double check the size of the gel you are preparing. All volumes and weights are given for a 50 mL gel.

1. Weigh 0.4 grams of agarose powder and place it in a 125 or 250 mL flask.
   
   
2. Add 50 mL of 1X TBE buffer to the flask. Swirl to mix the solution.
   
   
3. Place the flask in the microwave. Heat on high until the solution is completely clear and no small floating particles are visible (about 2 minutes). Swirl the flask frequently to mix the solution and prevent the agarose from burning.
   
   • Do not allow the agarose to boil over.
   • Use hot mitts when handling the flask because it will be very hot.
     
     
4. Cool the solution to 55 C before pouring the gel into the plastic casting tray. Higher temperatures will melt the plastic tray.
   
   
5. While the mixture cools, tape the ends of the gel tray with masking tape. Place the plastic comb in the slots on the side of the gel tray. The comb teeth should not touch the bottom of the tray.
   
   
6. Pour the agarose mixture into the gel tray until the comb teeth are immersed about 6 mm or 1/4" into the agarose. Push any bubbles to the side farthest from the wells.
   
   
7. Allow the agarose gel to cool until solidified. The gel will appear a cloudy white color and will feel cool to the touch (about 20 minutes). Gels can be stored, wrapped in plastic wrap, in the refrigerator for a few days.

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.