Protocol: Visualizing DNA in Agarose Gel using Ethidium Bromide – Post-electrophoresis Method

Ethidium Bromide is a DNA staining dye and can be used to visualize DNA in agarose gel. Both precast (Ethidium Bromide is added in molten agarose, just before casting the gel) and post-electrophoresis methods (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:

  • More accurate size determination than precast method

Disadvantages:

  • Time consuming and require extra an extra step
  • Consume more ethidium bromide than precast and preloading method
  • DNA can be visualized only after the electrophoresis is over

PROCEDURE

Precautions: Ethidium bromide is a known carcinogen. Take all necessary precautions while handling it.

Step 1: Prepare agarose gel of appropriate concentration and size in electrophoresis buffer.
For example, to prepare 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. 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 and fill the right amount of electrophoresis buffer (use the same electrophoresis buffer, TAE in this case) in the tank. 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, stain the gel with ethidium bromide solution
Once the gel run is over, turn off the power supply, and disconnect the wires. Take out the gel and submerge in an ethidium bromide solution (final concentration 0.5µg/ml in water or electrophoresis buffer). Incubate for 30 min with gentle shaking. 

After incubation, wash the gel with water 3- 5 times.

Step 4: Analyze the gel in the transilluminator or Gel Doc system.

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