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Agarose gel electrophoresis uses a porous agarose polymer network. For linear double-stranded DNA within a suitable separation range, shorter fragments generally move farther in a fixed run. Raising the agarose concentration changes the mesh and the range of sizes that can be resolved.^[ref] A compact circular DNA molecule need not migrate like linear DNA of the same length: geometry affects passage through the mesh.^[ref]

A concrete laboratory example is Addgene's documented DNA separation protocol. Its standard 1% gel contains 1 g of agarose in 100 mL of TAE buffer. Dissolved agarose sets in a casting tray around a comb; removing the comb leaves sample wells. The gel is covered with the same buffer solution in a box connected to a voltage source. Loading buffer contains glycerol to increase sample density and a tracking dye to show run progress. A DNA ladder occupies a separate lane beside the samples. With wells near the negative cathode, DNA migrates toward the positive anode. After separation, bound stain makes the DNA visible by fluorescence under a UV transilluminator. The DNA ladder supplies the size reference; the tracking dye monitors migration but does not identify sample DNA.^[ref]

In the manufacturer's controlled comparison, the same DNA ladder runs in 1%, 2%, and 3% agarose gels with the same run time and other stated conditions. The changed gel band spacing shows why a calibration belongs to its particular gel conditions, rather than establishing a universal distance law.^[ref]

In interpreting an unknown sample, a blank lane tests for detectable background or carryover in that blank, while the DNA ladder checks migration and detection of its known fragments. An empty sample lane cannot by itself distinguish absent DNA from unsuccessful loading or insufficient detection. These controls answer different questions; neither makes matching migration a proof of matching nucleotide sequence.

A DNA ladder contains DNA fragments of known lengths in base pairs. Its gel bands supply size references. Match the sample's strandedness and shape, and use the specified lengths for that DNA ladder. A size reference need not provide a quantitative mass standard.^[ref]

A gel band is a localized population of stained or otherwise detected molecules with sufficiently similar migration in one sample lane. One visible gel band need not contain only one nucleotide sequence. Two different linear DNA fragments of 375 base pairs can occupy the same unresolved gel band; equal migration supplies size evidence under suitable conditions, not a sequence determination.

Fluorescence from a bound stain can support an estimate of DNA quantity, but suitable staining and detector response matter.^[ref]

For a quantitative model with equal staining response per unit mass, background-subtracted, integrated fluorescence $I$ satisfies $I=km$. This requires an unsaturated detector and a common proportionality constant $k$; visual peak brightness alone does not supply $I$.

The amount of substance is $n=m/M$, where $M$ is molar mass. For double-stranded DNA with approximately equal average mass per base pair, $M$ is proportional to length $L$, so $n$ is proportional to $I/L$. In a constructed example, equal integrated signals from 250-base pair and 1000-base pair gel bands imply approximately equal masses but four times as many short DNA molecules. Conversely, equal numbers of those DNA molecules would give about four times the signal for the longer fragments in this model. Neither conclusion follows from darkness in a schematic drawing.