Agarose Gel Electrophoresis of DNA: Quantitative Biophysical Study of Mobility and Size Dependence

Abstract

Separation of complex mixtures of biomolecules via agarose gel electrophoresis is a cornerstone molecular technique. Yet quantitative data on electrophoretic parameters, including migration velocity and mobility across a wide range of DNA fragment sizes and structures, remain scarce in the primary literature. In this work, we present detailed biophysical analysis of DNA fragment migration in 1% agarose gel under defined experimental parameters (90 V, 80 minutes, 1× TBE buffer). As a size standard, 1 kbp DNA ladder was used to compute migration distances from UV-illuminated gel images using ImageJ software, and to determine electrophoretic velocities and mobilities for 13 fragments ranging from 250 to 10,000 bp within the ladder. Three samples of double-stranded DNA that included 750 bp, 2.7 kbp, 4.4 kbp (pBR322-BstNI digest) were examined. Also, circular single-strand and linear double-strand DNA of the bacteriophage φX174 DNA were examined. The migration velocity was inversely exponential with size over the 250-10,000 bp size range (R² = 0.99), decreasing from 8.87 x 10⁻⁴ to 2.69 x 10⁻⁴ cm/sec. The range for electrophoretic mobility (μ) was from 0.25 × 10⁻⁴ to 0.829 × 10⁻⁴ cm²/V·s. Circular ssDNA φX174 with μ of 0.249 × 10⁻⁴ cm²/V·s migrated significantly faster than the linear dsDNA form with μ of 0.162 × 10⁻⁴ cm²/V·s, even though the molecular weight of both is identical. This result reveals the strong influence of DNA structure on migration. These data agree with established electrokinetic theory and provide calibration references for DNA sizing in molecular biology and biophysics laboratories.