Steponeplus real time quantitative pcr instrument

Hit compounds (100 μM) were pre-incubated with the ABL N32L WT protein (1 μM) for 30 minutes in bicine assay buffer (10 mM bicine, 150 mM NaCl, pH 8.0). SYPRO Orange (Sigma) was added at 5X final concentration and fluorimetry profiles were acquired with a StepOnePlus real-time quantitative PCR instrument (Applied Biosystems) and software (version 2.3). Assays were performed in duplicate in sealed MicroAmp Fast 96-well qPCR plates (Applied Biosystems), and control reactions without proteins were included to correct for background fluorescence. Assays were equilibrated at 25°C for 2 minutes, followed by an increase in temperature at the rate of 1% (1.6°C/min) to 99°C, with continuous data collection. Mean fluorescence intensities, after subtracting background fluorescence, were plotted against temperature. Non-linear regression analysis using the Boltzmann sigmoid function in GraphPad Prism 6 was used to determine the T_m values, the midpoint of the melt curve between the minimum and maximum fluorescence intensities.

We used the TaqPath™ 1-Step RT-qPCR assay (Thermo Fisher Scientific, Waltham, MA) on the StepOnePlus Real Time Quantitative PCR instrument (Applied Biosystems, Foster City, CA) as an additional validation test on the contrived samples. Briefly, RNA samples were reverse transcribed to cDNA and then subjected to 45 cycles of quantitative PCR according to the following recommended conditions: UNG incubation at 25 °C for 2 min, reverse transcription incubation at 50 °C for 15 min, enzyme activation at 95 °C for 2 min, 45 cycles of amplification consisting of denaturation at 95 °C for 3 s followed by annealing/extension at 60 °C for 30 s, and a final infinite holding step at 4 °C. We used previously described primers (CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel) and 6-carboxyfluorescein (FAM)-labeled hydrolysis probes targeting three regions of the SARS-CoV-2 nucleocapsid protein (N) gene [27 ]. We also used additional primer probe set targeted a human housekeeping (RPP30) gene as an internal/extraction control. The primers and probes used for both the SARS-CoV-2 targets and the human RPP30 targets were identical to those described in the CDC nCoV-19 assay [27 ].