Part:BBa_K3001028:Experience

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Applications of BBa_K3001028

RNA Mango has been used as a fluorescent tag for tracking small non-coding RNAs (Autour et al., 2018) and live cell imaging (Dolgosheina et al., 2014). Here, we used RNA Mango to look at transcription occurring in real time. The RNA Mango template DNA was mixed with the Promega T7 RiboMax Express kit and incubated with thiazole orange (TOI) in a cuvette. The fluorescence intensity was recorded using a Quanta Master 60 fluorescence spectrometer (Photon Technology International). TOI was excited at 510 nm and the emission spectra recorded between 520 and 600 nm. Readings were taken as soon as the DNA template was added to the reaction, then at 5, 10, 20, 30, 40, 50 and 60 minutes (Figure 3A). As time progressed, the fluorescence intensity increased and then reached a maximum and leveled off. Peak fluorescence at 535 nm was then plotted against time (Figure 3B). The data was then fitted with a logistic growth curve (y = a / (1 + b e-kx ), k > 0) and was found to have a doubling time of 0.1355 min (~8 sec).

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