Gel Electrophoresis Protocol

Reagents:
Agarose
TAE or TBE Buffer at 1X Concentration
DNA or PCR sample
Ladder
Loading Dye
Equipment:
Gel casting apparatus (tray, base, and combs)
Power supply

Preparation

  1. Prepare a working solution of 1X TAE or TBE Buffer. Commercial stock solutions are often at a 10X concentration. To prepare 100 ml of a working solution of 1X Buffer mix the following in a sterile container:

10 ml of 10X stock solution + 90 ml Distilled Water

  1. Fit the gel casting tray into the base (with rubber gasket lining facing the walls of the base).

Making the Gel (For a 1% agarose gel in a 50ml volume gel apparatus)

  1. Weigh out 0.5 grams of agarose into a 250 mL conical flask. Add 50 mL of 1X Buffer. Swirl to mix.
  2. Microwave for about 1 minute to dissolve the agarose.
  3. The agarose solution can boil over very easily so continue to check it. Depending on the power of your microwave, it may be a good idea to pause it after 45 seconds and give it a swirl. Warning: the solution will be HOT. Wear protective gloves and hold it at arms length. If it is not completely dissolved, continue microwaving it for short periods of time, being careful to not allow it to boil over.
  4. Once the agarose is completely dissolved (solution is clear), place the flask on the counter and allow it to cool to approximately 55- 60°C.
  5. Pour the gel slowly into the tank. Push any bubbles away to the side using a disposable tip. Select the desired comb size and insert it into the corresponding slots of the gel casting tray.
  6. Leave the gel to set for at least 30 minutes, preferably 1 hour, with the lid on if possible.

Loading the Gel

  1. After the gel is set, carefully remove the gel comb. Lift the casting tray and reorient it 90 degrees so that the wells formed by the comb are at the top or positive end of the gel apparatus.
  2. Pour 1X buffer into the gel tank to submerge the gel to 2–5 mm depth. This is the running buffer.
  3. Thaw your DNA or PCR samples and loading dye if necessary. For each sample, pipet 1 μl of loading dye onto parafilm or into a sterile well of a PCR plate. Then, pipet 5 μl of sample and mix with the 1μl of loading dye.
  4. Transfer each sample mixture to the next empty well in the gel. Add 5 μl of ladder to an empty well. Be sure to take notes on which samples are in which wells.
  5. Close the gel apparatus by securing the lid in place. Switch on the power source and run it at 5 volts/centimeter. Therefore, if the distance between the electrodes is 10 centimeters, run the gel at 50 volts.
  6. Monitor the progress of the gel by reference to the marker dye. Stop the gel when samples have run approximately ¾ the length of the gel.
  7. To prevent electrocution, never touch the running buffer while the power supply is connected. FIRST, switch off power supply, THEN remove the lid of the gel apparatus and remove the gel.

Staining the Gel

Prepare and dilute your staining solution according to the product information. Be sure to follow ALL safety storage, handling, and disposal guidelines.

If using ethidium bromide, SYBR Green, or SYBR Safe staining solutions:

  1. Place the gel directly into a plastic staining container and cover entirely with solution.
  2. Allow the gel to sit in dark for approximately 30 minutes. Agitate as required.
  3. Transfer gel to a UV transilluminator or use Dark Reader lamp and glasses to view bands.

If using Fast Blast DNA stain:

  1. Place the gel directly into a plastic staining container and cover entirely with solution.
  2. Stain for 2-3 minutes. Pour staining solution back into storage flask and reuse up to 7 times.
  3. Rinse gel: Transfer gel to large container and pour 500-700ml of clean, warm (40-55°C) tap water so that it covers the gel. Gently shake for 10 seconds.
  4. First Wash: Dispose of the rinse water and refill with another 500-700 ml of clean, warm (40-55°C) tap water. Gently rock or shake on a rocking platform for 5 minutes…or manually shake once every minute.
  5. Second Wash: Dispose of first wash water and repeat step 4.
  6. After 5-15 minutes, analyze stained gel for expected bands on a white light transilluminator.