Agarose gels are horizontally laid and are completely covered by running buffer; hence they are referred to as submarine gels. The agarose gels are prepared in a gel casting trays which are of different sizes. The small gel slab are used for quick check in which the resolution is not great and runs for 30 to 40 minutes. Big gels are used for good resolution or separation of different sized DNA fragments, which generally runs for longer time, 60 minutes or more.

Electrophoresis buffer

For the electrophoresis of DNA many buffers have been recommended. But the most commonly used are TAE (Tris-acetate-EDTA) and TEB (Tris-botate-EDTA). Due to the difference in ionic strength of these two buffers, DNA fragments will migrate at different rates. The gels must be prepared in the same buffer in which the gels are run i.e. either TAE or TBE buffer. A buffer not only provides ions to support the conductivity, but also establish a pH. If you use concentrated buffer, enough heat may generate to melt the gel. The working concentration of the buffer is 1X and stock is prepared for 50X.

Preparing a agarose gel

Agarose gels are prepared as percentage weight/volume solutions. Thus to prepare standard 1% agarose gel 1 gram of agarose is dissolve in 100 ml of buffer. For bigger gels just scale up the volume accordingly. Agarose do not dissolve in the buffer, rather it has to be melted by boiling which is typically done in a microwave oven. While melting agarose, care should be taken to prevent boil over. To visualize the DNA on agarose gel UV-fluorescent dye is required. Ethidium bromide is a florescent dye that intercalates between bases of nucleic acids and allows convenient detection of DNA fragments in agarose under UV light. After agarose has cooled to about 60°C, a final concentration of 0.5 µg/ml ethidium bromide is added to agarose solution before pouring the agarose into the gel tray which is sealed and come is positioned. The agarose solution is allowed to cool at room temperature for an hour to solidify gel. Comb is carefully removed from the solidified gel and seal of the gel tray is removed. The gel is placed in the buffer tank carefully with buffer completely submerge the gel (Fig. 1).

Figure 1: A. Agarose gel casting unit. B. Solidified agarose gel.