Pouring agarose gels
Purpose: To prepare gels for gel electrophoresis.
- gel box, gel tray, comb
all in gel room
- 1% agarose in 1X TAE
premade, premelted bottles available in Peltier incubator; 564D
- SYBR Safe
in gel room, upper right hand shelf
- 100 ml glass bottle or beaker
prewarmed in incubator or water bath for a few minutes
- 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 of appropriate width, size, and number into niche at end of the tray.
- Pour agarose solution, 35 ml for small gels or 70ml for large gels, into prewarmed bottle. The marks on the side of the bottle should be a sufficient guide.
- Add 0.2 μl of EtBr per 35 ml 1% agarose. EtBr is extremely carcinogenic - be careful with it. (Wear nitrile gloves; dispose of tips properly into EtBr waste container; don't expose any part of the pipette other than the tip).
- Note that our EtBr is currently diluted to an unknown (but low) concentration. More EtBr may need to be added to achieve the same signal.
- Swirl bottle or mix with pipette tip to mix in EtBr. Pour the mixture into the tray. Rinse out the bottle with hot water.
- Let agarose sit for ~15 minutes to solidify. After it has set a bit (enough so that you can walk with it), you can set it in the cold room to chill further and solidify completely, saving a few minutes.
- 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.
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 left to right.
The capacity of the 8 well, 1.5mm wide well is approximately 45 μL. The capacity of the 15 well, 1.5mm well is approximately 15 μL.
- 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 ~85 V for 1 hr 20 mins (voltage and time values can vary). Use the timer to enable automatic shutoff of your gel.
- Verify that bubbles are rising from the electrodes once you start your gel to ensure your gel is running properly.