Agarose gel electrophoresis
(→Procedure: added room number)
(→Materials: DNA samples not a link anymore)
|Line 29:||Line 29:|
Revision as of 10:27, 26 July 2005
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
- Ethidium Bromide (abbreviated EtBr)
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).
- 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.)
You're now ready to load DNA samples and run your gel. Start at step #6 below in the Knight lab protocol. If there is a differing protocol for the Endy lab, please add it here.
Casting agarose gels
We precast our gels.
- Add 300mL 1X TAE to a 500 mL bottle.
- Measure out sufficient agarose to cast either a 1% (3 g) or 1.5% (4.5 g) gel.
- Add the agarose to the TAE buffer in the 500 mL bottle.
- Swirl to mix.
- Microwave bottle with loosened cap on high until the gel starts to bubble and is transparent.
This generally takes just over two minutes for 300 mL. If you microwave too long, the gel will bubble over causing a big mess and you will need to start over.
- Remove from microwave and let cool by either sitting on bench top or adding stir bar and placing on stir plate.
The advantage of the stir plate is that, if you forget about your gel for a while, it is less likely to solidify accidentally.
If you are in a hurry, you can place the bottle in a beaker of room temperature water on the stir plate to speed the cooling process significantly.
- While gel is cooling, assemble casting trays and gel combs and verify that the trays are level.
- Once gel is cooled so that it can be touched comfortably with your gloved hand, add 15 μL Ethidium Bromide (final concentration of 0.5 μg/mL).
- Pour gel into casting trays.
The height of the gel will depend on how much you wish to load. Diagnostic gels can be reasonably shallow since typically 10 μL volumes are loaded. For gel purifications, the gel should be deeper to enable loading of large sample volumes.
- Let gels sit until they are solidified.
Gels are solid when they are cloudy in appearance and firm to the touch.
- Gels may be used immediately. Alternatively, gels may be individually sealed in 6 x 10 inch polyethylene bags, labelled with initials, date and percentage and stored at 4 °C.
It is a judgement call as to whether a gel is too old to be used. If it takes on a shrivelled appearance, don't use it. If there is lots of condensation on the bag, only use it if your intended experiment isn't critical.
Running agarose gels
- Prepared DNA ladder
- Precast gel with the appropriate percentage and well size/numbers for your samples (see above)
- 1X TAE
- Loading dye
- Ethidium Bromide (10 mg/mL stock)
- Take a gel from the 4°C fridge.
If the number of gels is getting low, cast more gels as described above.
- Place your gel in gel box.
- Add 1X TAE buffer to gel box such that buffer just covers the top of the gel.
- Remove comb.
- Add 10 μL ethidium bromide stock solution to the running buffer well near the positive terminal.
- Load 12 μL prepared ladder
Typically load laddder in left-most lane and sometimes right-most lane as well depending on whether you have the space.
- Use 2 μL loading dye per 10 μL of sample.
- 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.
- Run your gel at ~85 volts for 1 hr 20 mins. Use the timer to enable automatic shutoff of your gel.
If you are in a hurry the gel can be run faster at ~95 volts for less than an hour.
- Verify that bubbles are rising from the electrodes once you start your gel to ensure your gel is running properly.
Visualizing agarose gels
Note that this procedure is under development
- FluorChem 8800 gel imager
- Remove gel from gel box shaking gently to allow residual buffer to fall back into gel box.
- Place in middle of UV box inside gel imager (you can leave the gel in gel tray).
- Close the door and turn on reflective white light button.
- In gel imager software, click the "Acquire" button such that gel displays on screen.
- Adjust gel position on UV box so that the entire gel is within the frame.
- Close the door, turn off reflective white light and turn on transilluminating UV light.
- In gel imager software, click "Acquire image" button to capture gel image to the screen.
It is occasionally necessary to adjust exposure time to improved image.
- Increase filtering if bands are difficult to see.
- Annotate gel as necessary.
- Save a copy of gel picture in your user folder.
- Remove gel and dispose in ethidium bromide waste container.
- Wipe down UV box if necessary.
If you are getting unexpected bands on your gel you may want to look at the Common Agarose Gel Issues