This study developed an improved method for estimating plasmid and DNA fragment sizes using agarose gel electrophoresis. Researchers tested various regression approaches on plasmids ranging from 1.37 to 312 megadaltons (MDa) and restriction fragments from 2.2 to 14.2 MDa. The multiple regression method (MR), which regressed log₁₀ molecular size against both log₁₀ relative mobility and the reciprocal square root of relative mobility, provided superior accuracy compared to earlier log-log methods. The MR approach yielded a mean percentage error of only 3.0% ± 1.5% across all plasmids tested. This method was robust across different gel concentrations and electrical voltages, and plasmid supercoiling did not significantly affect mobility across different bacterial hosts. The authors demonstrated that using just two Escherichia coli strains—V517 (containing eight plasmids) and IR713 (containing one larger plasmid)—as size standards allowed accurate estimation of unknown plasmid sizes. This approach provides comparable precision to more labor-intensive methods like electron microscopy while being considerably simpler and faster, making it practical for routine molecular sizing work.

Key findings

• Multiple regression combining log₁₀ relative mobility and reciprocal square root of relative mobility achieved mean percentage error of 3.0% ± 1.5% across plasmids of 1.37-312 MDa, significantly outperforming traditional log-log methods
• The method remained accurate across different agarose gel concentrations (0.8% and 1.4%) and varying electrical voltages, demonstrating robustness under different electrophoresis conditions
• Plasmid supercoiling did not significantly vary across different bacterial hosts, allowing the regression equation to work reliably with plasmids from various Escherichia coli and Pseudomonas species
• Using only nine plasmid standards from two E. coli strains (V517 and IR713) provided sufficient calibration for accurate size estimation of unknown plasmids in the tested range
• The method achieved precision comparable to electron microscopy contour length measurements while being simpler and more rapid than existing gel electrophoresis approaches