Agarose Gel Electrophoresis for DNA Visualisation

Weigh out agarose: 0.4 g for a 1% gel in 40 mL TBE (small gel, ~8 wells) or 1.0 g for 100 mL (medium gel, ~15 wells). Use a clean scale.

Transfer agarose to a microwave-safe flask or bottle. Add 1X TBE buffer to the target volume (40 mL or 100 mL).

Microwave on medium power for 60 seconds, swirl gently (heat-resistant gloves), microwave another 30 seconds, swirl, continue in 20-second bursts until agarose is fully dissolved (solution is clear, no crystals visible). Do NOT overheat — superheated liquid can flash-boil when disturbed.

Cool the molten agarose to ~60°C (touchable on wrist, hot but not burning). Typically 5–10 minutes at room temperature, or 2 min in a cool water bath.

Add SYBR Safe to 1× final concentration (4 µL of 10,000× stock per 40 mL, or 10 µL per 100 mL). Swirl gently to mix. Avoid creating bubbles.

Pour the molten agarose into the pre-prepared gel casting tray with comb inserted. Ensure no leaks at the ends. Pop any bubbles with a pipette tip.

Allow gel to set for 20–30 minutes at room temperature until fully solid (opaque throughout, no visible movement when tilted).

Transfer the cast gel to the electrophoresis tank. Fill the tank with 1× TBE buffer until the gel is just submerged (~1 mm over the top).

Gently remove the comb by pulling straight up. Do not pull sideways — it tears wells.

Prepare samples: mix 5 µL of PCR product with 1 µL of 6× loading dye on a clean surface or in the tube. Use a new tip for each sample.

Load samples into wells using a fine pipette tip angled against the front of the well. Deliver slowly and steadily — the sample should sink into the well (the loading dye's glycerol makes it denser than buffer). Include a well with 5 µL of DNA ladder as a size reference.

Connect the power supply: red (+) to the side AWAY from wells (DNA migrates negative→positive, toward red). Black (–) to the side with wells. Set voltage to 100 V.

Run the gel for 25–40 minutes, or until the front tracking dye (bromophenol blue, purple) has migrated about two-thirds the length of the gel. For small amplicons, stop earlier (slower dye front); for large amplicons, stop later.

Turn off the power supply. Carefully lift the gel tray from the buffer — gel is fragile.

Transfer the gel to the blue-LED transilluminator. Place it flat; the bottom of the gel (slot side) should face the camera. Turn on the blue LED. Bands appear as green-yellow fluorescent stripes.

Photograph the gel with a phone camera through the amber filter, or with a dedicated gel documentation system. Ensure a ruler or the ladder is visible for size reference.

Record the result in LibreBiotech: Assay record with gel image attached, lane annotations, and band-size calls for each sample. Link back to the Process that generated the PCR products.