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Pouring agarose gels
Purpose: To prepare gels for gel electrophoresis.
- gel box, gel tray, comb
- 1% agarose in 1X TAE
premade bottle usually above gel station
- 50-100 ml glass flask or beaker
- Push gel tray down into pouring box. The rubber gasket ends of the tray should form a seal with the sides of the box.
- Place comb(s) of appropriate width, size, and tooth number into notch in tray (usually thin 10-tooth comb).
- Melt agarose solution in microwave, taking out to shake every 30s-1min (HOT--use autoclave gloves). 250ml takes about 90s. Make sure it's clear and completely melted.
- Pour agarose solution into flask (~30 ml for small diagnostic gels). The marks on the side should be a sufficient guide.
- Let agarose sit for ~20 minutes to solidify.
- When gel has solidified, remove the comb, lifting straight up. Lift the gel tray and rotate it 90°, so that the ends of the gel are now exposed to the ends of the gel box. Wells should be at left side when plugs are away from you.
Running agarose gels
- Add enough 1X TAE to fill the reservoirs at both ends of the gel box and cover the surface of the gel--the gel should be immersed. (There are bottles of 1X TAE in the gel room on the right side of the bench. If they're empty, 1X TAE is above the sink in 564D.)
- Load prepared ladder (+dye +loading buffer). Typically, the ladder solution is at 1 μg per 12 μl, but you can change the concentration or total mass. The mass of ladder is important to know if you need to quantify your bands by comparison with the ladder bands.
Load ladder in left-most lane and sometimes right-most lane if you want to and have the space.
- Use 2 μL loading dye per 10 μL of sample. Some lab members use dyed loading buffer; others use clear sucrose solution. The dye makes the sample easier to see when loading, but may interfere with seeing bands later.
- Load samples in known order.
- Place gel box cover on gel box such that your samples will run towards the positive, red electrode. Make sure that the cables from the cover are connected to the power supply correctly.
- Turn on the power supply and run your gel at ~90 V for 1 hour (voltage and time values can vary). Use the timer to enable automatic shutoff of your gel.
- Verify that bubbles are rising from the electrodes and that the dye is migrating in the correct direction once you start your gel to ensure your gel is running properly.
- Wear nitrile gloves for this process. EtBr is extremely carcinogenic - be careful with it. The outside of the container should be kept clean, so don't touch it after touching contaminated surfaces.
- Place the gel into the plastic tray. Carefully pour EtBr staining solution over it to cover. Cover with cardboard lid to prevent exposure to light.
- Stain for about 1 hour, depending on the age and concentration of the stain.
- Pour the staining solution back into its bottle using the funnel.
- Pour destaining solution over the gel, and rock it back and forth every few minutes. (We use the used-up TAE as a destaining solution.)
- Pour the destaining solution back into its bottle using the funnel.
- Take the gel to the imager up in the Polz lab.
- To make more EtBr solution, add X μl of EtBr per ml TAE. Dispose of tips properly into EtBr waste container; don't expose any part of the pipette other than the tip.
- More EtBr may need to be added to achieve the same signal.