qPCR was performed on the ViiA7 Real-Time PCR system (Applied Biosystems). The VIC-labeled RNaseP assay was purchased from Thermo Fisher Scientific and used as an internal PCR control. The AmpR-specific primers and hydrolysis probe were taken from Roberts et al., 2017 (link), and the mNG21-10-specific primers and hydrolysis probe were designed as follows:
The primers and hydrolysis probes were purchased from Thermo Fisher Scientific and IDT, respectively. The AmpR and mNG21-10 assays were prepared by mixing 18 μM of each primer with 5 μM of the hydrolysis probe. qPCR reactions consisted of 1 μL cell lysate, 0.5 μL RNaseP assay (Thermo Fisher Scientific), 0.5 μL mNG21-10 or AmpR assay, 5 μL QuantStudio 3D Digital PCR Master Mix V2 (Thermo Fisher Scientific), and 3 μL water for a final volume of 10 μL. The qPCR thermocycling conditions were carried out as per the manufacturer’s instructions for the QuantStudio 3D Digital PCR Master Mix V2. The data was analyzed using the ViiA7 software and clones that were positive for mNG21-10 and negative for AmpR were selected for further screening by PCR as described below.
The primers and hydrolysis probes were purchased from Thermo Fisher Scientific and IDT, respectively. The AmpR and mNG21-10 assays were prepared by mixing 18 μM of each primer with 5 μM of the hydrolysis probe. qPCR reactions consisted of 1 μL cell lysate, 0.5 μL RNaseP assay (Thermo Fisher Scientific), 0.5 μL mNG21-10 or AmpR assay, 5 μL QuantStudio 3D Digital PCR Master Mix V2 (Thermo Fisher Scientific), and 3 μL water for a final volume of 10 μL. The qPCR thermocycling conditions were carried out as per the manufacturer’s instructions for the QuantStudio 3D Digital PCR Master Mix V2. The data was analyzed using the ViiA7 software and clones that were positive for mNG21-10 and negative for AmpR were selected for further screening by PCR as described below.
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