Ethidium bromide is a DNA staining dye and can be used to visualize DNA embedded in agarose gel. Both precast (ethidium bromide is added in molten agarose, just before casting the gel) and post-electrophoresis staining method (after electrophoresis, agarose gel is submerged in dye solution to stain DNA) work well with ethidium bromide but the preloading method (dye is added in DNA sample and then loaded onto the wells of agarose gel) do not give optimal results.
Advantages of the precast method:
- Relatively less ethidium bromide is required than post-staining method
- Relatively easy and time-saving
- DNA can be visualized any time during electrophoresis by exposing gel with UV light
Disadvantages of the precast method:
- Not good for size-based analysis of DNA fragments (ethidium bromide are reported to affect DNA mobility)
- Contaminate the Electrophoresis tank
Step 1: Prepare ethidium bromide-containing agarose gel of appropriate concentration and size in electrophoresis buffer.
For example, to prepare ethidium bromide-containing 0.8 % agarose gel of size – 10 cm x 12 cm x 0.4 cm, you require ≈50 ml agarose solution that can be prepared by dissolving 0.4 g agarose in a 50 ml electrophoresis buffer (e.g., TAE).
Weigh out 0.4 g agarose in a conical flask, add 50 ml 1x TAE buffer. Dissolve the agarose by heating the agarose suspension. Cool down the agarose solution to 50°C and add 2.5µl of ethidium bromide stock solution (Stock concentration: 10 mg/ml) and mix by gently agitating the solution. Pour the agarose solution into the casting tray and set the comb at the appropriate position. Wait until agarose is solidified properly (may take ≈ 30 min)
For more detail, please follow the instructions given in “Preparation of Agarose Gel for DNA Analysis”
Step 2: Load your DNA samples in agarose gel and start the electrophoresis.
Place the gel in the electrophoresis tank. Fill it with the right amount of electrophoresis buffer (use the same electrophoresis buffer, TAE in this case). Mix DNA samples with DNA loading dye and load them onto the wells of agarose gel. Place the tank cover, and connect the tank to the power supply. Run it at constant voltage between 70 – 100V.
For more detail, please follow the instructions given in “Running DNA Samples in Agarose Gel”.
Step 3: Once the gel run is over, turn off the power supply, and disconnect the wires. Take out the gel from the electrophoresis chamber and analyze it under UV transilluminator or Gel documentation system.