Estimating the Concentration of DNA by Fluorometry Using Hoechst 33258

Measuring the concentration of DNA using fluorometry is more sensitive than spectrophotometry, allowing the detection of nanogram quantities of DNA. In this assay, DNA preparations of known and unknown concentrations are incubated with the fluorochrome Hoechst 33258. Absorption values for the unknown sample are compared with those observed for a known dilution series, and the concentration of the unknown sample is estimated by interpolation. The assay can only be used to measure the concentration of DNAs whose sizes exceed ∼1 kb, as Hoechst 33258 binds poorly to smaller DNAs.