Varioskan flash spectral scanning reader

Prior to the fluorescence measurement, 2.5 μL of 10 × SAP buffer (NEB, China) and 2 μL of shrimp alkaline phosphatase (0.5 U/μL) were added to the PCR products and incubated at 37 °C for 20 min to degrade the unreacted Fl-dNTP. Following incubation, the mixture was held at 4 °C. For the fluorescence detection based on the fluorescence resonance energy transfer (FRET) ratio, 80 μL of 25 mM HEPES buffer, 20 μL of 15 μM PFP and 20 μL of PCR products were added to 96-well microtiter plates (Thermo Scientific). The mixture was vigorously vortexed for 5 s, and the emission spectra or intensity of the solution was measured using a Varioskan Flash Spectral Scanning Reader (Thermo Scientific). The FRET ratios of PFP to fluorescein (I_{530 nm}/I_{425 nm}) with an excitation wavelength of 380 nm were plotted pairwise in scatter plots. For the visible detection of FRET, 20 μL of PFP was added to the 96-well PCR plate containing PCR products and mixed thoroughly by pipetting. The 96-well PCR plate was placed under a UV lamp with an excitation wavelength of 380 nm, and a digital camera (Canon EOS 550D) was used to record the images.

For fluorescence detection using the fluorescence resonance energy transfer (FRET) ratio, 80 μL of 25 mM HEPES buffer and 20 μL of 15 μmol·L⁻¹ PFP were added to the 96-well microtiter plates (Thermo Scientific). The mixture was vortexed vigorously for 5 s, and the emission spectra or the intensity of the solution was measured by a Varioskan Flash Spectral Scanning Reader (Thermo Scientific) equipped with an excitation filter of 380/5 nm. Upon the addition of 20 μL of SBE products, the spectrum was measured again. The emission filters were 440/5 nm for PFP and 530/5 nm for fluorescein. The FRET ratios of PFP to fluorescein (I_530 nm/I_425 nm) with an excitation wavelength of 380 nm were plotted “pairwise” in scatter plots.
For visible detection of FRET, 20 μL of PFP was added to PCR tubes or the 96-well PCR plate containing the single-base primer-extension products and mixed thoroughly by pipetting. Photographs were taken in a WD-9403F UV Viewing Cabinet under 365 nm UV light irradiation, and a digital camera (Pentax k7) was used to record the images. The contrast of the images was adjusted so that the inherent fluorescence of the PCR plate was not visible. For the imaging of the 96-well PCR plate, it was better to position the 96-well PCR plate upside down to obtain uniform UV light irradiation on the SBE product/CCP mixture.