AGAROSE GEL ELECTROPHORESIS

Agarose gel electrophoresis is a method used in molecular biology to separate DNA strands by size, and to determine the size of the separated strands by comparison to strands of known length. It operates by a mechanism similar to sifting molecules through a sieve; an electric field is used to drag the negatively charged DNA molecules through a gel matrix, and the shorter DNA molecules move faster than the longer ones since they are able to slip through the gel more easily.

Material

For an agarose gel electrophoresis, several items are needed:

• The DNA that is to be separated.
• A DNA ladder, a mixture of DNA fragments (usually 10-20) of known size. The size of the DNA strands that are separated is determined by comparison of their relative position to that of the DNA strands of the DNA ladder. There are several DNA ladder mixes commercially available.
• TBE Buffer, 0.5x, pH 8.0
• EDTA, 0.5M, pH 8.0
• Agarose
• Ethidium bromide (5.25 mg/ml in H₂O)
• A color marker, i.e. a low molecular weight dye such as "bromophenol blue", to enable tracking the progress of the electrophoresis.
• A gel rack
• A "comb" (usually cut from a sheet of teflon)

Preparation

1. Make a 1% agarose solution in 0.5x TBE. If you analyze small DNA strands, go up to 2%. Use 15-70 ml, depending on the size of the gel.
2. Boil solution, preferably in a microwave oven.
3. Let the solution cool down to about 60°C at room temperature. Stir the solution while cooling.
4. Add 1 ml ethidium bromide on each 10 ml gel solution. Wear double gloves or nitrile gloves from here on, ethidium bromide is a mutagen! Some researchers prefer not to add ethidium bromide to the gel itself, instead soaking the gel in an ethidium bromide solution after running.
5. Stir the solution to disperse the ethidium bromide, then fill it into the gel rack.
6. Insert the comb at one side of the gel, about 5-10 mm from the border of the gel.
7. When the gel has cooled down and become solid, remove the comb. The holes that remain in the gel are the slots.
8. Put the gel, together with the rack, into a chamber with 0.5x TBE. Make sure the gel is completely covered with TBE, and that the slots are at the electrode that will have the negative current.
9. Add the color marker to the DNA. The DNA ladder is usually already stained.

Procedure

Inject the DNA ladder and the DNA into a slot each. Don't use more DNA solution than the slot can hold, usually 25 ml. Apply the electric current (usually 100 V for 30 minutes for a 15 ml gel). When the colored "front wave" reaches the end of the gel, stop the current. The color may leave the gel.

Figure 1 : Schematic drawing of the electrophoresis process. (Image in the PD).

(1) The agarose gel with three slots (S).

(2) Injection of DNA ladder into the first slot.

(3) DNA ladder injected. Injection of samples into the second and third slot.

(4) A current is applied. The DNA moves toward the positive anode due to the negative charges on its phosphate backbone.

(5) Small DNA strands move fast, large DNA strands move slowly through the gel. The DNA is not normally visible during this process, so the marker dye is added to the DNA to avoid the DNA being run entirely off the gel. The marker dye has a low molecular weight, and migrates faster than the DNA, so as long as the marker has not run past the end of the gel, the DNA will still be in the gel.

(6) The DNA is spread over the whole gel. The electrophoresis process is finished.

Illuminate the gel with an ultraviolet lamp (usually by placing it on a light box) to view the DNA bands - ethidium bromide fluoresces pink in the presence of DNA. Wear protective glasses! The DNA band can also be cut out of the gel, and can then be dissolved to retrieve the purified DNA.

See also: SDS-polyacrylamide gel electrophoresis