Agarose Gel Electrophoresis

 

Materials:

	agarose solution in TBE or TAE (generally 0.7-1%)
	1X TBE or TAE (same buffer as in agarose)
	gel loading dye
	10 mg/ml ethidium bromide

 

1. To prepare 100 ml of a 0.7% agarose solution, measure 0.7 g agarose into a glass beaker or flask and add 100 ml 1X TBE or TAE.
2. Microwave or stir on a hot plate until agarose is dissolved and solution is clear.
3. Allow solution to cool to about 55EC before pouring. (Ethidium bromide can be added at this point to a concentration of 0.5 μg/ml)
4. Prepare gel tray by sealing ends with tape or other custom-made dam.
5. Place comb in gel tray about 1 inch from one end of the tray and position the comb vertically such that the teeth are about 1-2 mm above the surface of the tray
6. Pour 50EC gel solution into tray to a depth of about 5 mm. Allow gel to solidify about 20 minutes at room temperature.
7. To run, gently remove the comb, place tray in electrophoresis chamber, and cover (just until wells are submerged) with electrophoresis buffer (the same buffer used to prepare the agarose)
8. Excess agarose can be stored at room temperature and remelted in a microwave.
9. To prepare samples for electrophoresis, add 1 μl of 6x gel loading dye for every 5 μl of DNA solution. Mix well. Load 5-12 μl of DNA per well (for minigel).
10. Electrophorese at 50-150 volts until dye markers have migrated an appropriate distance, depending on the size of DNA to be visualized.
11. If the gel was not stained with ethidium during the run, stain the gel in 0.5 μg/ml ethidium bromide until the DNA has taken up the dye and is visible under short wave UV light, if the DNA will not be used further, or with a hand-held long-wave UV light if the DNA is to be cut out and purified.