Agarose gel electrophoresis protocol (BioRad)

Materials

• BioRad electrophoresis supplies
  • PowerPac Power supply
  • Mini-Sub Cell GT Cell
  • Comb
  • Clear gel tray
• Water bath set at 60°C
• 1xTAE Buffer
• Agarose powder
  • Melt in 1x TAE buffer before the class; 1% (g/ml for this lab).
• SYBR safe
• DNA marker and samples
• Gel imaging system

Procedure

Assemble apparatus

1. Put the gel-casting tray into the electrophoresis box with the slots closer to the negative (black) electrode
2. Seal the gel-casting tray by sliding the gel-casting dams down the V-shaped grooves on both sides of the gel-casting tray
   • Ensure the gel box is completely sealed to prevent agarose leakage
3. Insert the comb into the comb-positioning slots closest to the negative (black) electrode

Cast gel

1. Obtain 30 ml of melted agarose with SYBR safe and immediately pour into the gel-casting tray
   • Act quickly to avoid gel solidification
   • Use a micropipette tip to push any air bubbles to the bottom of the gel
2. Let the agarose cool down and solidify, this may take about 15-20 minutes
   • Avoid disturbing the gel after pouring
   • Ensure the gel is completely solidified before proceeding to the next step
3. Pull the comb straight up
   • Avoid wiggling the comb to protect the wells
   • Rinse the comb and the gel dams with H2O and place them on paper towels to dry
4. Remove the gel-casting dams
5. Pour 1x TAE buffer to submerge the gel and the wells but not fill the gel box to the top

Load samples

1. Obtain the DNA marker and the PCR products from the instruction team
2. Determine the loading order of the samples on the gel and record the order in the notebook
3. Load 10 μL of each PCR sample and the DNA marker into the wells in the order you recorded
   • Avoid moving the gel box after loading the samples

Run gel

1. Gently place the cover on the gel box without disturbing the buffer or well loads
2. Turn on the power supply; set the voltage at 120 V
3. Push the “Running” button to start the gel running
   • Bubbles will form
   • Dyes will be visible in blue and yellow and will move toward the positive electrode.
4. Set the timer for 30-35 min
5. Turn off the power supply and remove the gel cover from the gel box
6. Lift the gel-casting box and slightly tilt it to let the buffer flow back to the gel box
7. Transfer the gel and the gel-casting box into a clear plastic tray
8. Carry the gel to the gel doc for imaging