Team:HKUST/Protocols/Agarose gel

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Agarose gel preparation and gel electrophoresis

• Purpose: To check the result
• Materials: Agarose, 0.5X TBE, SYBR safe DNA gel stain, DNA loading dye, DNA ladder/marker
  • Choice of ladders: VC100bp Plus DNA ladder (highest band 3000bp); Lamda DNA BstEII marker (highest band 8400bp, 1% argarose)
• Procedure:
  • To make a 0.8% agarose gel, use 0.16 g agarose per 20 mL 0.5x TBE.
  • Cover the flask with plastic wrap to prevent boiling over, then microwave the solution for 1-2 minutes to dissolve the agarose.
  • Let agarose solution cool for 5 minutes, then add SYBR safe DNA gel stain to a final concentration of 0.5 μg/mL.
  • Pour the agarose solution into a casting tray with well comb in place. Allow 20-30 minutes to completely solidify.
  • Place the agarose gel into the gel box and fill the box with 0.5X TBE until the gel is immersed.
  • Load an appropriate molecular weight ladder into the first lane of the gel. If needed, an additional ladder with different molecular weight could be loaded into the last lane.
  • Add 1 μL loading dye per 5 μL of sample.
  • Load the samples from left to right.
  • Cover the gel box and plug in the electrodes in a way that the samples will run towards the positive, red electrode.
  • Run the gel at 100V until the dye line is approximately 50-75% of the way down the gel. Approximately 20-30 minutes.
  • Carefully remove the gel from the gel box and check the result under UV exposure.
• Tips:
  • Higher concentration of agarose solution makes better resolution for less molecular weight expected band.
  • Let bottom of the flask be immersed in a cup of cold water for faster cooling.
  • In order for clearer band to cut specific band on the gel, immerse the gel in Ethidium bromide for 10-15 minutes after step k.
• Safety tips:
  • Be sure to wear a glove before treating the hot flask.
  • Ethidium bromide is a strong mutagen. Wear a lab coat, eye protection, and gloves when working with this chemical.