Real‐time velocity of DNA bands during field‐inversion gel electrophoresis

The velocity v of bands of double‐stranded, linear DNAs containing 48.5–5700 kbp was determined with 0.3 s resolution during field‐inversion agarose gel electrophoresis (FIGE) for a broad range of the forward pulse period T + , keeping the duration of the backward pulse T − = T + /3. Within 0.6 s or less after the field changed sign from − to +, the velocity showed a sharp positive peak; a similar spike, but with negative velocity, occurred immediately after the field changed from + to −. For long pulses, the magnitude of this spike increased with M 0.36 , reaching ten times the steady‐state velocity for M = 5.7 kbp. After this spike, the velocity dipped to 55–75% of its value in a steady field, then increased to a small secondary peak before reaching a steady‐state plateau. The duration of the velocity trough, and the time of the small peak, increased as M 1 . For standard FIGE conditions (ratio of forward:reverse pulse duration, T + : T − = 3:1; equal forward and reverse field amplitudes, E + = E − ), the mobility μ = ∫ v d t over a complete cycle was a minimum when E + terminated at the end of the velocity trough. The minimum occurred because the velocity during E + sampled primarily the trough, and because the backward velocity during E − was exceptionally large; the negative velocity spike was maximized when T + terminated at the end of the velocity trough. Computer simulations of FIGE by Zimm ( J. Chem. Phys. 1991, 94 , 2187–2206) and by Duke and Viovy ( J. Chem. Phys. 1992, 96 , 8552–8563) generate real‐time velocities that are in excellent agreement with our experimental data.