Equipment Requirements for Agarose Electrophoresis

Ray Gladden
Biotechnology Department

One of the most common questions asked by customers is "What type of equipment do I need to get started?" Equipment is required for the following manipulations: micropipetting, electrophoresis, and visualization. Micropipetting, or dispensing microvolumes of DNA, is best done by using adjustable micropipettors with disposable tips. These are relatively expensive, ranging from $200-$300 each depending on the brand purchased. If these are out of your budget range, a good inexpensive alternative is the Wiretrol microcapillary pipet, with a disposable tip and a reusable plunger. This option is not only inexpensive but is also very accurate and fairly easy to use. For dispensing premeasured volumes of predigested DNA, use disposable plastic needlepoint pipets. Electrophoresis is the separation of DNA fragments into different sizes through a matrix of agarose or acrylamide inside an electrophoresis chamber. The chamber has two electrodes on opposite ends, which can be attached to a DC power supply. Gel chambers are available with single (J8-21-3668) and double (J8-21-3654) casting trays. The double casting tray enables you to run two gels at one time in the same gel box. An electrophoresis buffer fills the chamber and conducts the electricity between two electrodes. When current is applied, the negatively charged DNA migrates toward the positive electrode. The agarose gel acts as a sieve, allowing the smaller-sized fragments to migrate faster than the larger fragments, thus separating fragments by size.

The speed of electrophoresis is dependent on the size of the gel and the amount of voltage applied to the gel box by the power supply. The higher the voltage, the faster the migration of the fragments. Each gel box has a maximum optimal voltage range, and exceeding this range results in smearing of the DNA bands. Lower voltages generally give cleaner separation of bands. We offer several power supplies (see pages 18-19), allowing you considerable flexibility in performance and cost. After electrophoresis, view the DNA by staining with ethidium bromide, methylene blue, or Carolina Blue(TM) DNA stain. Ethidium bromide is the stain most commonly used by researchers because of its sensitivity to DNA and the speed of staining. Drawbacks include the cost involved for its visualization (it requires a UV light source), and its suspected carcinogenicity. If you wear rubber gloves and work in a sink, the low concentrations required for staining can be used safely. Methylene blue stain is less sensitive than ethidium bromide, but this can be compensated for by using higher concentrations of DNA. It is also a visible-light stain, which means UV light sources are not required. Many instructors purchase inexpensive white light boxes, or utilize an overhead projector protected by a sheet of acetate. Another method is to place a clear piece of acetate over the gel and trace the DNA band patterns. A disadvantage of methylene blue is the time required for staining and destaining the gel. An alternative to these two stains is the Carolina BLU(TM) staining system.