Gel electrophoresis is a foundational laboratory technique used to separate large biological molecules, such as DNA fragments or proteins, based on their size and electrical charge. This separation occurs as the molecules move through a porous gel matrix under the influence of an electric field. The entire process relies on the precise introduction of samples into the gel, a function managed by the comb. The comb is an accessory that ensures a clean, uniform starting point for every sample before the electric current is applied to begin molecular separation.
The Comb’s Physical Design
The comb is generally constructed from durable, temperature-resistant materials like plastic or Teflon, designed to withstand the heat of the molten gel solution. Its most distinctive features are its “teeth,” which act as templates for the sample lanes. The number of teeth directly determines the number of individual samples, or lanes, that can be analyzed on a single gel slab.
Variations in the comb’s design, particularly the thickness of the teeth, directly impact the resulting sample wells. Thicker combs create wells with a larger volume capacity, useful when researchers need to load a greater amount of sample material, often for subsequent DNA extraction.
Conversely, combs with thinner teeth produce smaller wells. These smaller wells are preferred for analytical gels because they yield sharper, more concentrated bands, improving the visual resolution of the separated molecules.
Creating the Sample Loading Wells
The primary purpose of the electrophoresis comb is to create the small, defined pockets that hold the biological samples before separation begins. This process is initiated after the gel matrix, typically agarose or polyacrylamide, has been prepared in its liquid, molten state. The comb is carefully positioned and inserted into the casting tray while the gel is still fluid.
As the hot gel cools, the polymer structure begins to solidify, forming a mesh-like matrix around the submerged teeth of the comb. The comb must remain perfectly still during this polymerization process to ensure the integrity of the surrounding gel structure. Once the gel has completely set, the comb is slowly and vertically lifted out of the matrix.
The removal of the comb leaves behind a row of empty, rectangular depressions, which are the sample loading wells. These wells are defined cavities within the solid gel structure that serve as reservoirs for the DNA or protein samples mixed with a dense loading buffer. The precise and uniform shape of these wells is fundamental to the success of the experiment, as any imperfection can compromise the separation quality.
Factors Influencing Well Quality
The quality of the wells formed by the comb is directly related to the final clarity and accuracy of the separation results. One common issue is the formation of air bubbles trapped beneath the comb’s teeth when the molten gel is poured. These bubbles create physical defects or irregularities in the well bottom, which may distort the electrical field. This distortion causes the sample to migrate unevenly, leading to skewed or “smiling” bands later in the process.
Another practical consideration is the depth of the comb’s insertion into the liquid gel solution. If the comb is inserted too shallowly, the resulting well will be very thin at the bottom, increasing the risk of the sample floating out or mixing between lanes during loading. Conversely, if the well is too deep, the sample will be overly diluted across a large volume, potentially leading to faint or indistinct bands.
The technique used to remove the comb after solidification is also a source of potential error. A jerky or rapid removal can cause the delicate gel matrix to tear or wrinkle, particularly at the edges of the wells. To prevent this damage, the comb is often removed slowly and with a slight rocking motion, sometimes after adding running buffer to lubricate the interface.
The Comb’s Role in Sample Migration
The wells created by the comb establish the crucial starting line for the separation of the biological samples. Once the gel is placed into the electrophoresis chamber and submerged in a conductive buffer, the wells are positioned at the negative electrode (cathode). This placement is necessary because most biological molecules, such as DNA, possess an overall negative charge and will migrate toward the positive electrode (anode).
The well structure ensures that all molecules within a single sample begin their journey at the same point and across a narrow, uniform front. This initial concentration is necessary for the formation of tight, distinct bands as the molecules move through the gel matrix. If the sample were to start dispersed or unevenly, the resulting bands would be smeared or blurry, making it difficult to accurately compare the sizes of the separated molecules.