Gel electrophoresis is the standard lab procedure for separating DNA by size (e.g., length in base pairs) for visualization and purification. Electrophoresis uses an electrical field to move the negatively charged DNA through an agarose gel matrix toward a positive electrode. Shorter DNA fragments migrate through the gel more quickly than longer ones. Last Update: Feb. see FAQs below.
The gel (1) is a jelly-like substance made from agarose, a sugar polymer extracted from seaweed. The gel is immersed in a buffer solution and has electrodes (2 / 3) on either side, creating an electrical field. The gel is cast with small pockets close to the negative electrode. These are called wells (4). On a molecular level, the gel is not solid, but contains many small pores. Because the DNA molecule has a negative charge, due to its chemical structure, when a voltage is applied, the DNA fragments are pulled towards the positive electrode. The speed at which the DNA fragments travel through the gel depends on their size: Small pieces travel quickly, large pieces travel slowly. After some time, the DNA fragments have been separated and their size can be analysed. The gel can be infused with a DNA stain, which will bind to the samples. Using the transilluminator, the samples can then be made to fluoresce, so that they become visible.
A typical gel picture looks like the picture above. From each well, the DNA fragments have travelled in their lane have been separated by their size. In the left lane, a DNA ladder has been used. The orange lid (1) seals the gel box when the voltage is applied and also functions as a filter for the transilluminator, to render the fluorescing DNA visible. The base (2) is used to cast and run the gel. It has the red positive electrode, and the black negative one. Be careful when touching the electrodes, as the platinum wire is very thin and fragile. Two rubber dams are used to create buffer zones around the electrodes during gel casting (3). There are also combs to create 9 and 12 well gels (4).
Tip combs are laboratory tools used primarily in the field of gel electrophoresis, a widely employed technique for separating and analyzing macromolecules like DNA, RNA, or proteins based on their size and charge. The main purpose of tip combs is to create wells or pockets in the gel, allowing the researcher to load samples for analysis. Gel electrophoresis involves the movement of charged molecules through a gel matrix under the influence of an electric field. The gel, typically made of agarose or polyacrylamide, acts as a sieve, slowing down the movement of larger molecules compared to smaller ones. Tip combs are instrumental in creating wells or pockets in the gel during the casting process. These wells serve as slots for loading samples. The comb is placed at one end of the gel mold before the gel solidifies. After the gel has solidified, the tip comb is gently removed, leaving a series of wells in the gel. Researchers can then load their samples into these wells, usually containing DNA, RNA, or proteins. The wells created by tip combs determine the formation of lanes in the gel. Each lane represents a separate track along which the molecules will migrate during electrophoresis. Once the samples are loaded, an electric current is applied to the gel. Molecules move through the gel matrix based on their size and charge, resulting in separation. After electrophoresis, the gel is typically stained or treated to visualize the separated molecules. Tip combs come in various configurations, with the number and spacing of teeth determining the number and size of wells. In summary, tip combs are indispensable tools in gel electrophoresis, providing a structured and organized way to load samples for separation and analysis.
Why the Comb (Wells) Matter for DNA Separation
The wells created by the comb establish discrete lanes where DNA samples migrate during electrophoresis. Proper well spacing and number directly influence the resolution of fragment separation and the ability to compare multiple samples against a DNA ladder. As the DNA migrates toward the positive electrode, smaller fragments travel further down the gel while larger fragments stay higher, yielding a visible ladder of bands that can be analyzed to determine fragment sizes.
Practical steps tied to the comb and wells
- Cast the gel with the comb in place to form wells and lanes. Remove the comb after solidification to expose the loading pockets.
- Load samples carefully into wells, maintaining positive pressure to prevent bubbles and ensure sample stays in the well.
- Run the gel under an electric field, observing the separation of fragments by size through the formed lanes.
For visualization, a DNA stain can be integrated into the gel or buffer, and the stained DNA can be observed using a transilluminator or the orange lid as a filter. When documenting results, imaging should consider light conditions to maximize band visibility, often aided by using a darkened surface or an orange filter with a smartphone.
In typical protocols, buffers like 0.5X TBE or similar are prepared to maintain ionic strength and pH during the run. The buffer facilitates current flow and helps keep DNA soluble during separation. For agarose gels, a standard percentage (around 1%) is chosen based on the expected fragment sizes, with adjustments made to improve resolution as needed. Always wear gloves when handling gels and stains due to potential toxicity, even if many kits use relatively safe stains. After the run, the gel can be discarded safely following proper handling practices.
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Gel Electrophoresis
Key parameters to control include voltage, run time, and gel composition. A typical run might set the voltage to a moderate level (e.g., 50 V for compact systems) for tens of minutes, balancing resolution and thermal considerations. The DNA ladder serves as a size reference, enabling inference of fragment sizes in sample lanes. For enhanced resolution, consider adjusting gel percentage, loading volumes, or run duration, and ensure the wells are loaded evenly to avoid skewed results.
In summary, the comb creates the essential wells that define lanes, enabling the organized loading of samples and subsequent separation of DNA fragments by size under an electric field. The resulting banding pattern, viewed under fluorescence or filtered light, provides a direct readout of fragment sizes and sample composition.