Home > Agarose gel electrophoresis

1 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)
• Nitrile gloves
• A color marker, 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)

2 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 nitrile gloves(doubled for increased safety) from here on, ethidium bromide is a mutagenIn biology, a mutagen ( Latin, literally origin of change is an agent that changes the genetic information (usually DNA) of an organism and thus increases the number of mutations above the natural background level. Mutagens are usually chemical compounds! 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.

3 Procedure

Steps:

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 anodeAn anode is the positive electrode in an eletrolytic system or circuit. Literally, the path through which the electrons ascend out of an electrolyte solution. In electrochemistry, the anode is where oxidation occurs and is also the negative discharge plat due to the negative charges on its phosphateIn chemistry, a phosphate is a polyatomic ion or radical consisting of one phosphorus atom and four oxygen. In the ionic form, it carries a -3 formal charge, and is denoted PO3-. In a biochemical setting, a free phosphate ion in solution is called inorgan 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 ultravioletNote: Ultraviolet is also the name of a 1998 UK television miniseries about vampires. Ultraviolet UV radiation is electromagnetic radiation of a wavelength shorter than that of visible light, but longer than that of soft X-rays. It can be subdivided into lamp (usually by placing it on a light box) to view the DNA bands - ethidium bromide fluorescesultraviolet light in vials containing various sized cadmium selenide (CdSe) quantum dots. Fluorescence is a luminescence, i. optical phenomenon in cold bodies, in which a molecule absorbs a high-energy photon, and re-emits it as a lower-energy (longer-wav 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