Cfrench:AGE

Materials required
'''20 x TAE (Tris-acetate-EDTA buffer). '''
 * Dissolve 48.4 g Tris base and 3.72 g sodium-EDTA in about 450 ml water. Add 11.4 ml glacial acetic acid (fw 60.05, d 1.049). Acetic acid is corrosive and volatile, with a strong odour. Wear gloves and eye protection and work in a fume hood when handling glacial acetic acid. I suggest using a P5000 pipette to transfer 3 x 3.8 ml acetic acid. Rinse the pipette tip before disposal. Make up the final volume of the solution to 500 ml with water.

Agarose gel loading buffer
 * In a 1.5 ml microcentrifuge tube mix 0.6 ml 50% v/v glycerol, 0.3 ml 20 x TAE, and 0.1 ml 10% w/v SDS (see miniprep procedure for SDS recipe). Add a few grains of bromphenol blue. Mix by inversion. This is usually stored at 4˚C with the 1 kb DNA ladder, but is also quite stable at room temperature.

1 kb DNA ladder
 * This is provided by various manufacturers. We currently use the ladder from New England Biolabs. To make up 100 microlitres of this, mix 20 microlitres of the stock solution (stored at -20˚C), 60 microlitres of water, and 20 microlitres of loading buffer in a 1.5 ml microcentrifuge tube. This can be stored at 4˚C with the loading buffer.

Procedure
Note: this procedure is for a 30 ml 0.8% w/v agarose gel. Amounts of agarose and buffer must be adjusted for other types of gel. The procedure described is for a Bio-Rad mini sub-cell GT electrophoresis tank, but other tanks for horizontal (submarine) electrophoresis are basically similar.


 * 1. Insert the buffer dams tightly in the gel tank, add the comb and place at 4˚C to cool. For general purposes, use the thin 8 well comb.


 * 2. Weigh 240 mg agarose and add to a 100 ml conical flask.


 * 3. Measure 13.5 ml 20 x TAE in a 25 ml measuring cylinder. Pour it into a 500 ml measuring cylinder and make it up to 270 ml with de-ionised water. Pour this into a 500 ml beaker and swirl to mix. Note: this makes 1 x TAE. The procedure will work equally well with 0.5 x TAE, and may even be better in some cases, since the gel will run less hot.


 * 4. Measure 30 ml of 1 x TAE into the 100 ml conical flask using a 100 ml measuring cylinder. Put the rest of the 1 x TAE aside for now.


 * 5. Tare the conical flask with TAE and agarose on the 400 g balance.


 * 6. Place the flask in the microwave together with a beaker of water (this is a powerful microwave, and some extra water should always be included when less than 50 ml liquid is being mcirowaved). Microwave on medium power for 1 minute. Swirl to mix. Microwave on medium power for a further minute. Swirl and oberve. If the solution si clear, it is ready to use; if there are still visible particles in it. microwave on medium power for a further 30 seconds.


 * 7. Place the flask on the tared balance and carefully add water drop by drop, while swirling, to make back up to the original weight (to replace water that evaporated during the microwaving process).


 * 8. Cool the flask to about 55˚C (just hot enough to be almost too hot too hold through a latex glove). You can do this by swirling it in room temperature water (waterbath if switched off, otherwise under the cold tap) for about 30 seconds.


 * 9. Remove the gel tank from the refrigerator. Pour a small amount of gel into the tank and swirl it around the edges until it sets. This will form a seal and prevent leakage. Pour in the rest of the molten agarose. Make sure the combi is sitting correctly in ints notches. Leave for about ten minutes or more to set.


 * 10. Remove the buffer dams and comb. Pour the remaining TAE into the ends of the tank to cover the gel. The gel is now ready to load.


 * 11. Load samples in suitable buffer into the wells. If the thin 8 well comb was used, each well can hold up to 15 microlitres or so.


 * 12. Set the power pack to 100 V, 50 mA (for a 30 ml gel) and run for about 40 minutes (or less if less separation is required). Check after a minute or so to make sure the dye is migrating in the right direction.

Staining the gel
Gels can be stained with either ethidium bromide or SYBR-Safe. Ethidium bromide is cheaper but more toxic, and must be visualised under ultraviolet light, which damages the DNA (this is important if you are planning to elute the DNA from the gel). SYBR-Safe is more expensive but non-toxic, and can be visualised under blue light which is not harmful to the eyes and does not damage DNA (though ultraviolet illumination can also be used). '''For safety reasons, use of ethidium bromide is being phased out in this department. Use of SYBR-Safe is recommended.'''

Staining with ethidium bromide
An ethidium bromide stock solution (10 mg/ml in water, I think) is in the toxics cabinet. When fresh stain is required, add 20 microlitres of this to 100 ml water in a plastic tub with a sealable lid. '''Safety note: ethidium bromide is toxic and mutagenic and is a suspected carcinogen! Wear gloves when handling ethidium bromide solutions!''' Soak the gel in this for 20 minutes. Transfer the gel to a small plastic tray, rinse with water (discarding the water into a large beaker) and visualise under ultraviolet illumination. '''Safety note: ultraviolet radiation is harmful to the skin and eyes! Wear gloves and a face shield when using an exposed ultraviolet source!'''

If bands are very faint, you can restain the gel for longer, perhaps adding more ethidium bromide stock solution. If the gel background is high, you can destain the gel for 20 minutes or so in water; the ethidium bromide that is not bound to the DNA will diffuse out of the gel, reducing the background intensity and making bands easier to see. Dispose of all contaminated solutions safely as described below.

When the procedure is complete, discard the gel into the large beaker, add sufficient water to cover it completely, and add a destaining bag. Leave this overnight, then discard the gel into the autoclave waste (wrapped in a latex glove). The water can be disposed of to the drain. The destaining bag can be reused several times. It should then be placed in the waste container in the large toxics cabinet for safe disposal.

The ethidium bromide stain can be reused several times. If it starts to lose its effectiveness, further ethidium bromide stock solution (5 microlitres or so at a time) can be added. When the solution is to be discarded, it should be placed in a glass beaker with a destaining bag as described above, and left overnight. If there is obvious microbial growth in the solution (using the acetate which leaches out of the gels), bleach can also be added; otherwise, the solution should be transferred to a glass bottle and put out for disposal by autoclave.

Staining with SYBR-Safe
SYBR-Safe (Invitrogen) is one of several non-toxic substitutes for ethidium bromide that have recently become available. It is supplied as a 10 000 x stock solution in dimethyl sulfoxide, which is stored in the refrigerator. Note: DMSO freezes at refrigerator temperatures. Take the solution at and allow it to thaw some time before you want to use it. DMSO is readily absorbed through the skin. Wear gloves when handling solutions in DMSO. To use SYBR-Safe, add 1 microlitre of stock solution to 50 ml water in a small plastic tray. Immerse the gel in this for 20 minutes or so.

The gel can be visualised under blue light (Invitrogen Safe Imager) or ultraviolet. For use of ultraviolet, see safety notes above. The stain can be stored in a bottle in the refrigerator and reused several times. When no longer useful, it can be disposed of via the drains.

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