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using other instruments such as Xuorescent microplate readers [6], microplate Xuorometers [7,8], and transillumi-nator–microplate–CCD camera systems [9], but the instru-ments might not be readily available in most molecular biology laboratories. Real-time PCR instruments are common in molecular biology laboratories and can be used to measure Xuores-cence. DNA intercalated with a dye emits Xuorescence at 520 nm under excitation at 490 nm, and with real-time PCR instrumentation the assay is carried out in PCR tubes, equivalent to spectrophotometer cuvettes used with a Xuo-tured at 95 °C and the annealing process is slow and incom-plete, the tubes for collecting the well factor need to be extra tubes (we used extra blank tubes with EvaGreen but without DNA) rather than the experimental tubes them-selves. When the temperature reached 25 °C, the well factor tubes were replaced by experimental tubes, including blank ones, and the intensity of Xuorescence was measured. The user-deWned part of the running program set for the assay was 100 cycles of 25 °C for 5 s, 25 °C for 1 s, and 25 °C for 10 s. Although we used 100 cycles, more cycles could be used if necessary. The program was paused during the Wrst 5 s at 25 °C to replace PCR tubes, and Xuorescence wasAnalytical Biochemistry x