Gel electrophoresis is a fundamental laboratory technique used to separate deoxyribonucleic acid (DNA) fragments based on their size and electrical charge. This separation process causes the DNA to move through a gel matrix when an electric current is applied, resulting in distinct lines called bands. To make sense of these bands and determine the actual length of the unknown DNA fragments in a sample, a reference material is always required. This material, run in a separate lane alongside the samples, is known as the DNA ladder, or molecular weight marker, and it acts as the necessary scale for accurate analysis.
The Purpose and Structure of a DNA Ladder
The DNA ladder serves as a molecular ruler, providing a standard for comparison to estimate the size of unknown DNA molecules after they have been separated. Without this size standard, the separated bands would show only that different sizes of DNA are present, but not what the specific lengths are. The ladder itself is a prepared solution containing a mixture of multiple double-stranded DNA fragments of known, precise lengths.
These known lengths are measured in base pairs (bp) or kilobase pairs (kb), with one kilobase pair equaling 1,000 base pairs. When the ladder is loaded into a well on the gel and subjected to the electric current, its constituent fragments separate just like the sample DNA. Each band visible in the ladder lane corresponds to a specific, pre-determined base pair length.
The largest fragments remain closer to the well where the sample was loaded, while the smallest fragments migrate the farthest down the gel. This composition ensures that the ladder is affected by the same conditions, such as the gel concentration and the electrical field, as the unknown DNA samples.
Interpreting the Bands: Sizing Unknown Samples
Reading the results of the gel begins with understanding the inverse relationship between the size of a DNA fragment and the distance it travels. Shorter DNA fragments move more easily through the pores of the gel matrix and therefore travel further from the well. Conversely, longer, heavier fragments are hindered by the gel and remain closer to the starting point.
To determine the size of an unknown fragment, a direct visual comparison can be made between the sample band and the bands in the adjacent ladder lane. If your sample band aligns horizontally with a ladder band, the unknown fragment is approximately the same size as that known ladder fragment. For example, if a sample band stops exactly at the same point as the ladder’s 500 bp band, the sample fragment is estimated to be 500 base pairs long.
More often, a sample band will fall somewhere between two bands of the DNA ladder, requiring a more precise estimation. In this case, the size of the unknown fragment is interpolated by judging its position relative to the two nearest known sizes. For instance, if a sample band is halfway between the 400 bp and 500 bp ladder bands, it can be estimated to be around 450 bp.
For the most accurate determination, especially in research settings, a standard curve is constructed. This involves measuring the exact distance each ladder band traveled from the well and plotting this distance against the logarithm of its known base pair length. This plot typically yields a relatively straight line, allowing for a precise mathematical calculation of the unknown fragment’s size based on its measured migration distance.
Visualizing Results and Common Ladder Types
The DNA ladder, like the sample DNA, is not visible until the gel is stained with a fluorescent dye (such as ethidium bromide) and placed under ultraviolet (UV) light, which causes the bands to glow. The size range of the unknown samples dictates which type of ladder is selected for the experiment.
Ladders are generally categorized by the range of fragment sizes they contain, such as a 100 bp ladder, which is suitable for smaller fragments up to about 1,500 bp, or a 1 kb (1,000 bp) ladder, which covers a much wider range, often up to 10,000 bp or more. Researchers select the ladder that best brackets the expected size of their target DNA fragment.
To aid in quick identification and orientation, most commercial DNA ladders include one or two reference bands. These specific bands are engineered to be either brighter or thicker than the other bands in the ladder. For example, in many 1 kb ladders, the 1,000 bp and 3,000 bp fragments are often enhanced for immediate recognition on the gel.