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Measurement of Molecular Beacons in the SpectraMax Gemini Spectrofluorometer (MaxLine Application Note #36)

ere Nunc brand, black 384well microplates, surface-treated to minimize binding (cell culture-treated). Allsamples were prepared in 10 mM Tris buffer, pH 8, containing 1 mM MgCl2, and the final concentrations of beacon and target were 0.3 M and 1.6 M respectively, in a total volume of 54 L. The reaction mixtures were incubated at least 30 minutes at ambient temperature before measurement in a SPECTRAmax GEMINI microplate spectrofluorometer.


RESULTS AND DISCUSSION
Figure 1 shows the fluorescence spectrum of a tetrachlorofluorescein (TET)labelled probe in the presence and absence of complementary target. At the peak maximum (approximately 535 nm), the probe/target complex had approximately 35 times higher signal than did the probe alone.


The optimal wavelength settings for TET and several other molecular beacons were determined by examination of signal/blank ratios as previously described.5,6 The combination of excitation/emission wavelengths and emission cutoff filters giving the highest ratios are summarized in the table below.


There is considerable interest in measuring multiple targets (multiplexing) in a single sample. This can be done by using a different fluorophore for each probe, assuming the probes do not interfere with each other. If their emission spectra do not overlap, detection of one in the presence of the other is straightforward. Such is the case for fluorescein (FAM)- and tetramethylrhodamine (TAMRA)-labelled probes. Using the instrument settings intended for FAM, the TAMRA probe/ target complex does not interfere with the FAM signal ( Figure 2 ).


Conversely, when TAMRA settings are used, the FAM probe/target complex does not interfere with the TAMRA signal (Figure 3). There are many other 2-probe combinations which would be compatible. If two probes have partially overlapping em
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