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. An electrical current pulls the molecules through a porous gel matrix, causing smaller fragments to travel farther and faster than larger ones. Since the molecules are colorless and invisible within the translucent gel, researchers cannot analyze their results. This problem is solved by the specialized equipment known as the UV box.
Defining the UV Transilluminator
The UV box is formally called a UV transilluminator, a specialized piece of equipment. This transilluminator provides a high-intensity source of ultraviolet (UV) light that shines upward through a glass surface where the gel is placed. The UV light emitted is typically in the mid-range of the UV spectrum, often around 302 nanometers, which is suited for exciting common fluorescent dyes. Some transilluminators also offer shortwave (around 254 nm) or longwave (around 365 nm) UV settings, or even visible blue light, depending on the specific dye being used. The primary function of the transilluminator is to provide the necessary energy to make the invisible molecules observable for analysis.
The Necessity of Fluorescent Dyes
The UV transilluminator requires a partnership with a fluorescent dye to visualize the molecules. These dyes are chemical compounds that bind specifically to the target molecules, such as DNA or RNA. Common examples include ethidium bromide (EtBr) or safer alternatives like SYBR Green or GelRed.
When the dye is bound to the nucleic acid, the complex absorbs the high-energy UV radiation from the transilluminator. This absorption excites the electrons within the dye molecule to a higher energy state. As the excited electrons relax back to their ground state, they release the absorbed energy in the form of light. This emitted light has a longer wavelength than the UV light absorbed, placing it in the visible spectrum, often appearing bright orange or green. The dye-molecule complex illuminates only the regions of the gel where the separated DNA or RNA fragments are located.
Interpreting the Visible Bands
The final result of the transilluminator’s illumination is the appearance of distinct, bright lines, or “bands,” on the gel. Each band represents a collection of molecules, like DNA fragments, that are roughly the same size. The relative position of a band on the gel indicates the size of the molecule, with bands closer to the starting point (the wells) containing larger fragments and those farther away containing smaller fragments.
To accurately determine the size of the sample fragments, a DNA marker or “ladder” is run alongside the samples. This ladder contains fragments of known lengths, providing a reference scale against which the sample bands can be compared. Additionally, the brightness or intensity of a band gives an indication of the relative concentration of the molecule present. Once the bands are visible, researchers use a specialized camera system to capture a permanent image of the illuminated gel for later analysis.
Working with the UV transilluminator requires following safety precautions due to the intense UV radiation it produces. Exposure to this high-energy light can cause damage to skin and eyes, including conditions similar to sunburn or photokeratitis. Users must always wear personal protective equipment, such as a UV-blocking face shield, and ensure the transilluminator’s built-in protective cover is correctly in place when the light source is active.