Gel electrophoresis is a laboratory technique used to separate large biological molecules, such as DNA, RNA, and proteins, based on their size and electrical charge. This separation is achieved by moving the molecules through a porous gel matrix under the influence of an electric field. The technique is fundamental in molecular biology for analyzing nucleic acids and proteins in applications ranging from diagnostics to research. Preparing for a successful run requires a specific collection of equipment and chemical reagents.
Essential Electrophoresis Hardware
The physical structure needed begins with the gel electrophoresis chamber, often called the gel box or tank. This container holds the gel and the conductive buffer solution, providing a contained environment for the separation. The chamber includes two electrodes, typically color-coded red for the anode (positive charge) and black for the cathode (negative charge), which conduct the electrical current. Since DNA and RNA are negatively charged, they are loaded near the cathode and migrate toward the anode.
A separate power supply unit is required to generate the electrical current necessary to drive the charged molecules through the gel matrix. This unit allows the user to set a constant voltage, current, or power level, maintaining a steady migration pace. Connecting the power supply to the gel box electrodes with lead cables establishes the electric field across the gel.
The gel itself must be formed using a specialized gel casting tray and a gel comb. The casting tray is a mold used to pour the molten gel material, allowing it to solidify into a slab. The gel comb is inserted into the liquid gel before it sets, creating small indentations called wells where the samples will be loaded. This hardware assembly forms the functional core of the electrophoresis system.
Materials for Gel Matrix Construction
The separation medium is constructed from specific materials chosen based on the type and size of the molecules being analyzed. For separating DNA and RNA fragments ranging from 50 to 20,000 base pairs, agarose, a natural polysaccharide derived from seaweed, is the standard material. Agarose is dissolved in a buffer solution, heated until clear, and then poured into the casting tray to solidify into a porous gel.
For separating proteins or very small DNA fragments (typically 5 to 500 base pairs), polyacrylamide is often used instead. It forms a gel with a more uniform and finer pore size, but requires careful handling as its liquid components are neurotoxins. The concentration of the matrix material (agarose percentage or polyacrylamide concentration) directly controls the size of the pores, which determines the resolution of the separation.
The gel matrix must be run submerged in an electrophoresis buffer, a salt-containing solution that maintains a stable pH and conducts the electrical current. Two common choices for nucleic acid separation are Tris-Acetate-EDTA (TAE) and Tris-Borate-EDTA (TBE). TBE is preferred for sharper resolution of smaller DNA fragments (under 2 kilobases), while TAE is better suited for separating larger fragments or when subsequent enzymatic reactions are planned. The EDTA component in both buffers acts as a chelating agent to prevent the degradation of nucleic acid samples by metal-dependent enzymes.
Chemical Reagents for Sample Preparation and Running
Accurate sample handling demands specialized tools, notably micropipettes and disposable tips. Micropipettes allow for the precise measurement and transfer of extremely small liquid volumes (typically in the microliter range). This ensures that samples and reagents are mixed correctly and loaded accurately into the gel wells. Samples are usually prepared in small microcentrifuge tubes before loading.
A specialized chemical mixture called Loading Dye is added directly to the sample before loading onto the gel. This dye serves two main purposes: increasing the density of the sample and visually tracking its migration. The density agent, often glycerol or Ficoll, causes the sample solution to sink neatly into the well rather than diffusing into the buffer solution.
The colored tracking dyes within the mixture, such as Bromophenol Blue or Xylene Cyanol FF, do not stain the molecules but travel alongside them. These dyes provide a visual marker of the separation front. The operator watches these colored bands to know when to stop the electric current, preventing the samples from running off the end of the gel. The final component required is the sample itself, such as isolated DNA or protein, intended for separation and analysis.
Equipment for Visualization and Analysis
The separated molecules in the gel remain invisible to the naked eye, necessitating the use of specific stains and imaging equipment to make the results observable. The most common method involves a visualization stain, or intercalating agent, that binds directly to the DNA or RNA molecules. Ethidium Bromide (EtBr) has historically been the standard stain, but because it is a known mutagen, safer fluorescent alternatives are now widely utilized.
Modern stains like GelRed, GelGreen, or SYBR Safe are designed to be less hazardous because they are membrane-impermeant, meaning they cannot easily enter living cells. Once stained, the gel is placed on a UV transilluminator. This device emits shortwave light that excites the fluorescent dye bound to the nucleic acids, causing the DNA bands to glow brightly. A camera system or specialized gel documentation unit is then used to capture a permanent image of the fluorescent bands for record-keeping and analysis.