SensiFAST™ Probe No-ROX One-Step Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Prepare standard 20 uL final reaction mix. Reverse transcribe at 45°C for 10 min, initiate polymerase activation at 95°C for 2min	If PCR efficiency below 90% Increase extension time/Increase concentration of primers in 100nM increments

Protocol tips
Prepare standard 20 uL final reaction mix. Reverse transcribe at 45°C for 10 min, initiate polymerase activation at 95°C for 2min
Downstream tips
If PCR efficiency below 90% Increase extension time/Increase concentration of primers in 100nM increments

Publication protocol

DNA (200 ng) was extracted from ears, oral and anal swabs, and 50 ng of DNA was extracted from sera and used for testing. The real-time PCR was performed using SensiFast probe no ROX kit according to supplier recommendations (Bioline GmbH, Luckenwalde, Germany). Primers and probes are listed in Table 1. The reaction was performed in 20 μL. The PCR conditions were as follows: enzyme activation for 5 min at 95 °C was followed by 45 cycles of denaturation at 95 °C for 10 s, annealing at 59 °C for 20 s, and extension at 62 °C for 30 s. Design of the reference plasmid was described previously [24]. For the target quantification experiments were accompanied by a serial 10-fold dilution (1x105, 1x104, 1x103, 1x102, 1x101 and two or one copy) of the reference plasmid, example is shown (Figure 1). The sensitivity of the uniplex real-time PCR system was 1–2 copies per reaction and the estimated efficiency of the real-time PCR was between 0.98 and 1.02. The real-time PCR was performed as uniplex or as duplex PCR, using the porcine cyclophilin A gene as a housekeeping control with primers and probe described previously [25]. Reaction was performed in a Stratagene MX3005P system (Agilent Technologies, Santa Clara, CA, USA). Each sample was tested in duplicates, once using uniplex real-time PCR and once using duplex real-time PCR. In addition for confirmation, oral samples were tested using two uniplex real-time PCRs and one duplex real-time PCR. Serum samples were tested using two uniplex real-time PCRs and one duplex real-time PCR. Anal swabs were tested using three uniplex real-time PCRs. Ear biopsies were tested using two uniplex real-time PCRs and one duplex real-time PCR.

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Manufacturer protocol

Download the product protocol from Bioline for SensiFAST™ Probe No-ROX One-Step Kit below.

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