Agarose gel electrophoresis is a fundamental molecular biology technique used to separate and analyze deoxyribonucleic acid (DNA) fragments based on size. The method involves applying an electric field to move negatively charged DNA molecules through a porous, gel-like matrix. This process allows researchers to visualize, isolate, and estimate the size of DNA pieces.
The concentration of the agarose powder mixed with the buffer, expressed as a weight-per-volume percentage, is the primary factor determining the quality and range of DNA separation. Selecting the correct percentage optimizes the resolution for a specific range of fragment sizes. A poorly chosen concentration can result in DNA bands that are either too close together to distinguish or that run off the end of the gel.
Understanding the Sieving Mechanism
The process of DNA separation relies on the gel acting as a microscopic sieve, a network of channels that impede the movement of molecules. Agarose is a polysaccharide polymer that solidifies into a three-dimensional matrix when heated and cooled in a buffer solution. This polymerization creates a mesh structure with pores through which the DNA must travel to reach the positive electrode.
The concentration of agarose dictates the size of these pores, establishing an inverse relationship: a higher percentage creates a denser matrix with smaller pores. As DNA molecules are drawn through the gel by the electric field, larger fragments experience greater frictional resistance. They are forced to adopt a process known as “biased reptation,” where the molecule must temporarily orient itself to squeeze through the tight channels.
Smaller DNA fragments encounter less friction and move through the pores more freely, allowing them to migrate faster and farther than their larger counterparts. The ability of the gel to clearly distinguish between two DNA fragments of similar size (resolution) depends entirely on this sieving effect. Adjusting the agarose percentage changes the pore size to optimize this frictional difference for the fragment sizes of interest.
Matching Gel Percentage to DNA Size
The percentage of agarose determines the effective range of DNA fragments that can be separated and resolved clearly. Lower concentration gels contain large pores ideal for separating long DNA molecules, while higher concentrations are necessary for distinguishing between small fragments. The optimal percentage ensures the DNA fragments are spread across the length of the gel, maximizing the separation distance.
Large Fragments (5 to 50 kb)
For analyzing large DNA fragments (5 to 50 kilobase pairs [kb]), a low concentration of 0.5% to 0.8% agarose is recommended. This large-pore matrix allows the heavy fragments to migrate a sufficient distance into the gel.
Medium Fragments (500 bp to 10 kb)
When working with medium-sized DNA pieces, such as polymerase chain reaction (PCR) products or restriction digests ranging from 500 base pairs (bp) to 10 kb, a standard 1.0% to 1.5% gel is used.
Small Fragments (50 bp to 500 bp)
Separating very small fragments (50 bp to 500 bp) requires a high-percentage gel, often between 1.8% and 2.5%. The tight mesh provides the necessary resistance to slow down these small molecules, allowing for noticeable separation between fragments that differ by only a few base pairs. Concentrations higher than 2.5% are generally avoided, as the gel becomes brittle and the run time significantly increases.
Impact of Running Conditions
Beyond the agarose concentration, several external parameters influence DNA migration and the final quality of the separation. The choice of electrophoresis buffer is significant, with Tris-acetate-EDTA (TAE) and Tris-borate-EDTA (TBE) being the two most common options.
TAE buffer generally provides faster DNA migration and is preferred for separating large DNA fragments over 2 kb, as the acetate ions promote better resolution in this size range. TBE buffer utilizes borate ions and offers a higher buffering capacity, making it a better choice for long-duration or high-voltage runs because it maintains a stable pH longer. TBE also provides superior resolution for smaller DNA fragments, typically below 1000 bp, resulting in sharper, more defined bands.
The applied voltage is another variable, commonly expressed as volts per centimeter (V/cm) between the electrodes. Running the gel at a high voltage (over 10 V/cm) accelerates separation but generates excessive heat, which can cause DNA bands to become smeared or distorted. Conversely, very low voltages improve resolution but lead to prolonged run times, which can cause band broadening as the DNA diffuses. A voltage gradient between 4 V/cm and 8 V/cm is often a practical compromise, though very large DNA fragments are best separated using lower voltage gradients (sometimes below 5 V/cm) to prevent band smearing.