Standard neutral agarose electrophoresis

 

Standard neutral agarose electrophoresis

Standard agarose gels can be prepared using either TBE or TAE running buffers.

You will need:

Either 10 x TBE or 10 x TAE buffers
1 x TBE buffer - 89mM Tris, 89mM boric acid, 2mM EDTA, pH 8.
1 x TAE buffer - 40mM Tris-acetate, 2mM EDTA, pH 8.
10mg/ml ethidium bromide stock solution
10 x Type III loading buffer (50% glycerol, 0.5% bromophenol blue, 0.5% xylene cyanol, this solution should be stored at 4°C)

N.B: Extreme caution should be excersised when handling solutions containing ethidium bromide as it is a powerful mutagen. Gloves should always be worn. Solutions containing high levels (> ca. 10ug/ml) of ethidium bromide should be treated with a strong oxidizing agent (i.e. potassium permanganate) to render the ethidium bromide less dangerous before disposal into waste bottles for that purpose. Disposal of waste should be by a route capable of dealing with such reagents (i.e. a contracted company).

1) Agarose gels (0.6-2%) are prepared by dissolving powdered agarose in the required volume of the appropriate electrophoresis buffer using either a bunsen burner or a microwave oven.

2) Once completely dissolved, allow to cool to approximately 50-55°C, add sufficient 10mg/ml ethidium bromide to give ~0.5ug/ml and pour into the appropriate electrophoresis gel forming rig with the comb inserted.

3) Once set, remove the comb and transfer the gel to the appropriate gell apparatus. Cover the gel with the appropriate running buffer. For routine gels, ethidium bromide at a concentration of 0.5 ug/ml (from a 10mg/ml stock) should be incorporated into the gel running buffer.

4) Prior to loading DNA samples, 0.1 volumes of 10 x Type III loading buffer should be added.

5) Electrophoresis should be performed at voltage gradients of between 2 and 12 volts per centimetre depending on whether accurate sizing of fragments (2V/cm) or routine checks of plasmid concentration and yield (12V/cm) are required.

6) Continue electrophoresis until the slowest dye front (xylene cyanol FF) has migrated approximately half the length of the gel. This should give sufficient resolution for most purposes. The operators discretion should obviously be used here depending on the size of the DNA molecules of interest.

7) Visualize DNA by illumination of the gel using a 302nM UV transilluminator.

Molecular weight markers can be used to estimate the size of DNA fragments.