Use of Ethidium Bromide Fluorescence Enhancement to Detect Duplex DNA and DNA Bacteriophages during Zone Sedimentation in Sucrose Gradients: Molecular Weight of DNA as a Function of Sedimentation Rate

Duplex DNA molecules and DNA bacteriophages have been sedimented through 5-25% sucrose gradients containing ethidium bromide. The location of DNA within the gradients has been determined by illuminating gradients with ultraviolet light and observing the ethidium bromide fluorescence enhancement induced by the DNA. The relative sedimentation rates of linear, duplex DN As from bacteriophages T4, T5, T7 and an 8.3% T7 deletion mutant have been determined. The distances sedimented by DNA have been corrected, when necessary, for a progressive decrease in sedimentation rate that occurs after the DNA has traversed 40% of the sucrose gradient. The corrected distances sedimented by two DNA molecules, r1' and r2', are related to the DNA molecular weights, m1 and m2, by the equation: r1'/r2' = (m1/m2)0 38 when 0.025-0.70 μg of each type of DNA is sedimented. Intact bacteriophages were also sedimented in ethidium bromide-sucrose gradients and detected by fluorescence enhancement.