Agarose gels and Electrophoresis
Agarose gels are an easy, cheap and effective method of separating, and viewing, DNA. The agarose concentration of the gel you make is determined by the size of the DNA fragments you intend to resolve, and view. For the majority of DNA work, a gel of 1% agarose is fine and is good for DNA sizes that are between 400bp and 10kb (roughly) and where the two DNA fragments you want to resolve are not of a similar size. If they are similar sizes then you may have to change the concentration. For example, for two fragments of 500 and 700 bp a 2-3% gel would help to separate them. For two fragments that are 10-11kb you would probably need a 0.5% agarose gel, and run it for a long time at a slightly lower voltage (to prevent melting).
Gels can be made with either TAE or TBE buffers. There are differences between them that may, or may not, be important to you. See the notes and tips on the buffers page.
1) Add either autoclave tape or electrical tape to the ends of your gel setting tank to hold the gel in whilst setting. Don’t use masking tape as this leaks when wet. Then add your combs so it is ready for pouring. Remember to tape some of the wells of the comb together to make a bigger well if you want to gel extract a DNA fragment (which might mean loading up to 100 µl). Some tanks have rubber stoppers that fit on the end, if this is available it may be less messy than using tape.
2) Weigh the correct amount of agarose into a conical flask or 500ml bottle with a functioning lid and rim (see Tip 1). Most tanks have a recommended gel volume; usually between 40-120 ml. therefore use between 0.4-1.2 grams for the 1% gel.
3) Add the correct amount of buffer to the bottle or flask and mix by swirling.
4) If using a bottle, undo the lid slightly to allow steam to escape. Do not heat with the lid closed.
5) Heat the agarose solution in a microwave until it begins to bubble. Then remove and swirl to mix the solution. It will be very hot so use protective gloves.
6) Microwave until the agarose particles are dissolved. If you swirl the mixture and look at in the light you will see any un-dissolved agarose particles moving around.
7) If using a bottle you can immediately tighten the lid up when you take it from the microwave. Then if you run the bottle under cold water the gel liquid will begin to boil and bubble as it cools. This happens because you cool the air in the bottle which makes a vacuum and allows more efficient heat exchange between the liquid and the gas. Be careful not to cool it too much, you should just be able to hold it comfortably.
8) If using a conical flask then leave it to cool on the bench side until it is just comfortable to hold. Swirl occasionally to ensure the bottom doesn’t set first.
9) Add 1µl of Ethidium Bromide (Potential Hazard: ETBR is a potent carcinogen, wear cloves and dispose of tips and liquid properly) stock per ml of gel. Stock is at a concentration of 500 µg/ml ETBR in water. Final concentration of 0.5 µg/ml i.e. use one 1µl of ETBR stock per ml of gel. For a safer alternative, see note 1.
10) Swirl gently to mix but avoid getting bubbles.
11) Pour the gel from one corner slowly; don’t make it too thick as you might get a lot of auto fluorescence when you image it.
12) You should wait a little while before you use it (approx 40 minutes - 1 hour). It will become opaque when set.
13) The gel starts to set after 15 minutes, and if you are in a hurry, you may then place the gel in a fridge and wait only a further 15 minutes.
14) To run a sample, the gel is placed in an electrophoresis tank containing buffer so that the gel is just immersed (a few millimetres). The DNA will run from negative to positive, so ensure you place it in the correct orientiation.
15) Load your sample by placing your tip just inside the top of a well and slowly ejecting the sample. The glycerol (if homemade) or Ficoll (if pre-bought) in the loading dye will make the sample sink to the bottom.
16) Load a DNA ladder in an adjacent lane. Which one depends on the size ranges of your samples.
17) Run the gel at 10V/cm of gel.
18) Fragments are observed using UV light and a permanent photographic record is made if required.
19) Loading too much DNA can cause problems to separate the bands so try to avoid this is if possible.
Tip 1: Many lab bottles have a blue or clear plastic rim which can go missing or get burnt by flaming. These blue rims prevent the liquids running down the bottle when pouring and keep an air tight seal after autoclaving. Make sure the rim is intact.
Tip 2: Some companies sell low melt agarose which can help with making the gel. Remember that some gel runs, particularly in TAE, can cause excessive heat. This may damage your gel on extended high voltage runs.
Tip 3: Running gels at above 10 volts per gel cm length can cause the gel to melt.
Tip 4: Try to make your gel from the buffer you intend to run it in, if not, you may get a conductivity imbalance. Also, old buffers in tanks that have been repeatedly topped up can have a higher salt concentration than a gel made from the same buffer because of evaporation. This can also cause a conductivity imbalance and lead to poor gels.
Note 1: Using SYBR Safe DNA Gel Stain is a safer, non-carcinogenic alternative to ethidium bromide. We have not verified its activity.
Page written and produced by Dr Ryan Cawood