Agarose Gel Electrophoresis
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Materials:
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agarose solution in TBE or TAE (generally 0.7-1%) |
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1X TBE or TAE (same buffer as in agarose) |
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gel loading dye |
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10 mg/ml ethidium bromide |
- 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.
- Microwave or stir on a hot plate until agarose is dissolved and solution is clear.
- 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)
- Prepare gel tray by sealing ends with tape or other custom-made dam.
- 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
- Pour 50EC gel solution into tray to a depth of about 5 mm. Allow gel to solidify about 20 minutes at room temperature.
- 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)
- Excess agarose can be stored at room temperature and remelted in a microwave.
- 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).
- Electrophorese at 50-150 volts until dye markers have migrated an appropriate distance, depending on the size of DNA to be visualized.
- 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.
