EZQuant–ELN Knowledge Base

Agarose Gel Electrophoresis	Table of Contents

Gel preparation:

• Prepare an agarose gel solution by mixing agarose powder with electrophoresis buffer (usually TAE or TBE) to the desired concentration.
• Heat briefly by microwave to dissolve the powder. Do not allow the solution to boil.
• Cool to 50-60°C, add ethidium bromide to the gel (final concentration 0.5µg/ml)
  and pour into the gel tray.
  (Note: Ethidium bromide is a known mutagen and should be handled as a hazardous chemical - wear gloves while handling).
• Lower the comb gently into the molten gel.
• Allow the gel to solidify 30 minutes.

Sample and marker preparation:

• Prepare DNA marker: Mix 3µl of DNA marker stock solution, 22µl of ddH₂O and 5µl of 6x loading dye in a microcentrifuge tube.
• Add 4µl of 6x loading dye into 20µl of samples for gel electrophoresis (10-20ng DNA) and spin for 2 seconds in a microcentrifuge.

Electrophoresis:

• Put the solidified gel into the gel apparatus.
• Add TAE buffer to cover the gel and remove the comb.
• Load 10µl of each sample into the wells. Load 10µl of DNA marker.
• Run the gel at 100-150V for 20 minutes. Use 100V with mini-gel apparatus, and 150V with midi-gel apparatus.
• Move the gel onto UV light box (put on proper face shield and any other necessary gear to protect skin and eyes from UV).
• Take a Polaroid photo under UV.