In this lab, you will use agarose gels to separate DNA molecules produced in PCR reactions. These PCR products should be well-resolved on 1.25% agarose gels prepared in TAE buffer, which provide good separation of molecules that are smaller than 2 kb. Place the casting tray into the gel apparatus. If you are using the BioRad apparatuses, position the black wedges at each end of the casting tray.
1. Determine the amount of agarose that you will need for a 1.25% (1.25 g/100 mL) gel that fits your casting platform. Most of the gel apparatuses in the lab are the BioRad Mini-Sub GT systems with a 7 cm x 7 cm casting tray that accommodates a 30 mL gel. Check your calculations with your teammates before you proceed.
2. Fill a graduated cylinder with the appropriate volume of TAE buffer. Pour the solution into a small flask.
3. Weigh out the appropriate amount of agarose. Sprinkle the agarose onto the surface of the TAE in the flask. Note: the agarose will not dissolve until it is heated.
4. Dissolve the agarose by heating the solution for intervals of 15-20 seconds in a microwave oven. After each interval, remove the flask and gently swirl it around a bit to disperse the contents. Note if the agarose particles are still apparent or if the agarose has dissolved. The best gels are made from agarose that has NOT been overcooked.
SAFETY NOTE: The agarose solution will be very HOT when you remove it from the micro- wave! Use caution when handling the flask. Be particularly careful not to contact the steam that will be coming through the opening of the flask. Fold several paper towels and wrap them around the neck of the flask when you handle it. If you do happen to spill some hot agarose on your skin, wash it immediately with cold water and alert your TA.
5. Allow the agarose solution to cool until you can comfortably touch the flask with your hands. Agarose solutions over 60 ̊C will warp the casting tray! Pour the gel. Place the sample comb in place. Do not move the casting platform until the gel sets. You will know that the gel is set when it becomes opaque. Allow the gel to cure for about 20 minutes after it sets.