Gel electrophoresis is a technique used to separate biological molecules, such as DNA, RNA, or protein fragments, based on size and electrical charge. The method provides a way to estimate the size of these molecules. The principle relies on passing an electric current through a porous gel matrix, which acts as a molecular sieve. Since nucleic acids carry a net negative charge, they move toward the positive electrode. Smaller molecules move faster and farther through the gel’s mesh-like structure than larger ones.
Preparing the Gel Matrix
The gel matrix is typically made from agarose, a polysaccharide extracted from seaweed. The concentration of agarose powder is calculated based on the size of the DNA fragments being separated. Lower percentage gels (0.7% to 1.0%) create larger pores, suitable for fragments greater than 1,000 base pairs. Higher concentration gels (1.5% to 2.0%) yield a finer mesh necessary for resolving small fragments under 500 base pairs.
The agarose powder is mixed with a buffer solution, which acts as the electrolyte needed to conduct the electrical current. Common buffers include Tris-acetate-EDTA (TAE) and Tris-borate-EDTA (TBE). TAE is preferred for separating longer DNA fragments, while TBE offers better resolution for smaller fragments. The buffer used in the gel must also be used as the running buffer.
The mixture is heated, often in a microwave, until the agarose is completely dissolved and the solution is clear. Careful monitoring is necessary to prevent the solution from boiling over, which can change the concentration of the buffer. Once the solution has cooled slightly, it is poured into a casting tray where a comb is suspended near one end. As the solution cools, the agarose forms a solid, porous gel, and the comb molds the wells that will hold the DNA samples.
Assembling the Electrophoresis Unit
Once the gel solidifies and becomes firm and translucent, move the casting tray into the electrophoresis tank. Gently remove the comb, leaving the rectangular wells at one end of the gel. Take care not to tear the delicate agarose walls of the wells during removal.
Fill the tank with the prepared running buffer, ensuring the liquid fully submerges the gel. The wells must be positioned closest to the negative electrode, typically marked black. Since DNA is negatively charged, placing the samples near the negative electrode ensures they will be repelled and migrate toward the positive electrode, which is marked red.
Place the lid, which contains the electrode leads, securely onto the tank. Connect the leads to the power supply terminals, matching black to black and red to red, to establish the circuit. Confirm the setup is correct before turning on the power, as reversing the polarity would cause the DNA samples to run out of the wells.
Sample Preparation and Loading
The DNA sample must be mixed with a specialized gel loading dye before loading. This dye serves multiple functions. It contains a density agent, such as glycerol or Ficoll, which increases the density of the sample solution, allowing the sample to sink easily into the submerged wells without floating away. The dye also includes tracking dyes, such as Bromophenol Blue, which are visible and allow monitoring of the separation progress since DNA is invisible. Additionally, some dyes contain EDTA, which chelates divalent metal ions that could interfere with the experiment.
To load the sample, use a micropipette to draw up the mixture. Position the pipette tip directly over the well entrance and lower it just inside the opening, taking care not to puncture the gel bottom. Slowly dispense the sample into the well, relying on the density agent to pull the liquid down. Proper technique ensures the DNA migrates as a tight, uniform band.
Running the Separation
After loading all samples, including a DNA size marker, turn on the power supply to initiate separation. The voltage setting is a consideration; higher voltage speeds up the run but generates excessive heat. High heat can lead to “gel smiling,” where uneven heating distorts the separation. A common guideline is to set the power supply to about 5 volts per centimeter measured between the electrodes.
The current causes the negatively charged DNA fragments to migrate away from the negative electrode and toward the positive electrode. As the run progresses, the movement of the tracking dye is observed to monitor the separation front. For instance, Bromophenol Blue co-migrates with fragments around 300 base pairs in a 1% agarose gel, providing a rough estimate of fragment travel distance.
The separation continues until the tracking dye has migrated about two-thirds to three-quarters down the gel length. Stopping too soon results in poor separation, while running too long risks losing the smallest fragments off the end. Once complete, turn off the power and safely disconnect the electrical connections before removing the gel for visualization.