Agarose gel electrophoresis is a fundamental technique in molecular biology used to separate and analyze deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) fragments. The process separates nucleic acids based on their size by applying an electric current across a slab of gel. This allows researchers to quickly visualize the success of an experiment, estimate the size of a DNA product, and gauge the quality of a sample.
Understanding Band Migration and Staining
The separation of DNA fragments relies on the molecule’s natural negative charge, which is a result of the phosphate backbone. When an electric field is applied across the agarose gel, the negatively charged DNA molecules are drawn toward the positive electrode. The agarose acts as a porous matrix, impeding the movement of the DNA fragments as they travel through it.
The rate at which a DNA fragment moves is inversely proportional to its size. Smaller fragments weave through the pores more easily and travel further down the gel, while larger fragments encounter more resistance and remain closer to the starting wells. After separation, the DNA fragments concentrate into distinct, visible lines called “bands.”
To make these bands visible, a fluorescent dye, such as ethidium bromide, is added to the gel or running buffer. This dye binds to the DNA and fluoresces brightly when exposed to ultraviolet (UV) light, allowing the separated bands to be photographed and analyzed.
Using the DNA Ladder to Determine Size
Determining the length of an unknown DNA fragment requires the use of a DNA ladder, also known as a molecular weight marker. The ladder is a mixture of DNA fragments of known, specific sizes, which is loaded alongside the samples. These fragments separate during electrophoresis to form a distinct set of bands, each representing a known length, typically measured in base pairs (bp) or kilobases (kb).
To estimate the size of a sample band, its migration distance is directly compared to the bands in the adjacent ladder lane. If a sample band aligns horizontally with the 500 bp band on the ladder, the unknown fragment is approximately 500 bp long. For bands that fall between two ladder markers, researchers visually interpolate the distance to estimate the size, as this provides a quick and reliable estimation for routine analysis.
The selected ladder must span the expected size of the sample fragments to provide an accurate comparison. Since the distance DNA travels is inversely proportional to the log of its molecular weight, the bands on the ladder typically appear closer together at the higher molecular weight end.
Assessing Sample Quality and Concentration
The appearance of the bands provides qualitative information about the sample’s quality and relative concentration. DNA quality is assessed by the sharpness and discrete nature of the bands. A clean, sharp band indicates that the DNA molecules are intact and of a uniform length, suggesting a successful extraction or reaction.
Conversely, a “smear”—a diffused streak of fluorescence—suggests that the DNA has degraded into a multitude of random sizes. This degradation is often caused by enzymes or harsh handling.
The concentration of DNA in a band is estimated by its brightness or intensity; a visibly brighter band contains a greater amount of DNA than a faint band of a similar size. The brightness is directly related to the amount of fluorescent dye incorporated, which correlates with the total mass of the DNA.
While the gel offers a quick, relative measure of concentration by comparing the sample band’s intensity to the ladder, it is not a precise quantification tool. This visual assessment helps determine if there is enough material for downstream applications.
Interpreting Common Experimental Outcomes
The patterns observed on an agarose gel are direct evidence of an experiment’s success or failure. A successful polymerase chain reaction (PCR) or a clean plasmid preparation often results in a single, sharp band at the predicted size. This outcome confirms that the reaction amplified or isolated only the intended DNA sequence.
The appearance of multiple bands in a single lane typically indicates either the presence of multiple products or the outcome of a restriction enzyme digest designed to cut DNA at several locations. If no bands appear, it suggests a complete failure of the experiment, such as a lack of DNA amplification, or an error like failing to load the sample.
Additionally, a dense, high molecular weight smear of fluorescence remaining near the starting well often signifies the presence of unseparated, high-mass genomic DNA. This genomic DNA did not fully migrate into the gel matrix.