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Agarose gel electrophoresis Created 2026-10-14 Updated 2026-10-18
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]
Five Main Nucleic Acid Gel Electrophoresis Steps by Thermo Fisher Scientific. Step 1, Selection and gel preparation; Recommended percentage of agarose gels for separation of DNA fragments
A compact circular DNA molecule need not migrate like linear DNA of the same length: geometry affects passage through the mesh.[ref]Five Considerations for the Nucleic Acid Gel Electrophoresis Process by Thermo Fisher Scientific. Effects of conformation, Figure 1: nicked circular, linear and supercoiled forms of the same DNA
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]
Five Main Nucleic Acid Gel Electrophoresis Steps by Thermo Fisher Scientific. Step 1, Figure 3 and caption: same DNA ladder at three agarose percentages under the same conditions, including run time
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.