Maheshri:Gel

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Casting an agarose gel

1. Make 1x TAE buffer by diluting the 50X stock. You will need ~350mL for the small Bio-Rad gel box and ~1000mL for the medium Bio-Rad gel box.
2. Add agarose and 1x TAE buffer to a 250mL Erlenmeyer flask. Add sufficient agarose to 30-40mL of TAE for small gels or 80-100mL of TAE for medium gels. A 0.8% agarose gel is usually good for resolving DNA fragments of 0.5-1kb. Cast 1.5% or 2.0% if you need to resolve smaller fragments.
3. Microwave TAE and agarose 1 to melt. Be careful not to overboil. Take out the flask and let it cool down until you can touch the flask with bare hands. Add Ethidium bromide (EtBr) and swirl the flask to mix. The EtBr stock is 20000X so add 1μl per 20mL of agarose gel solution. Dispose the EtBr contaminated tip in the designated waste container located in the chemical fume hood.
4. Place the gel tray in the casting device. The two ends of the tray should be tightly sealed. Place combs of appropriate width and number into niche at the end of the tray.
5. Pour the mixture into a tray. Rinse out the flask with water immediately after pouring.
6. Let the gel sit for at least 15 minutes. After the gel has solidified, remove the combs, lift the gel tray and put it into the gel box.