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Agarose Gels to analyze DNA

1. Microwave 1% agarose in TAE with a refluxed flask. Swirl periodically to make sure the agarose melts uniformly and completely.
   • Higher percentages of agarose (1.5%-2%) are used to resolve smaller bands and lower concentrations.
2. Set up the gel tray with stoppers or tape to mold the agarose. Add the well comb.
3. Once the agarose has cooled slightly, pour gel into the bed enough to provide 2-3mm well depth. Allow 10-15 minutes to cool and solidify before proceeding.
4. Take off gel bed stoppers and tape, place in gell box and add enough 1xTAE to barely cover the gel. Gently remove the comb and check the wells for irregularities.
5. Add electrodes to the gel box remembering that the DNA should RUN TO RED
6. Load samples starting from the left. The first and/or last well should be a ladder of control DNA (e.g. 1kb, 1kb+)
7. Position the gel so that the wells are clearly visible, and supporting the pipette with your other hand, position the tip so that it is on top of the well and gently inject solution. The solution will sink to fill the well
8. Attach electrodes to the power supply, color coded. Turn the power dial down completely, and switch the supply on. Then, increase the power to the desired voltage (we use 100V/130V depending on length).
   • Alternately, if using a toggle power supply, turn on the power supply and select the voltage and time. Then, connect the leads and press start.
9. Once the tracking dye (part of the loading buffer) has reached close to the bottom of the gel, turn off the power, disconnect the electrodes, and with gloves on, grasp the gel box by the ends and lift out while draining excess TAE into the gel box
10. Slide the gel into the stain (EtBr) gently so that no splashing occurs. Turn the shaker on low until it is gently shaking. Stain for 10 minutes, and with gloves/spatula take the gel out and rinse in dH2O.
11. Image and photograph on the lightbox
    • Make sure to label wells and bands on the photograph with a marker/pen.