PerfeCTa MultiPlex qPCR SuperMix

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.	PCR plates, tubes and tips should be UV sterilized for 20-30 mins

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.
Protocol tips
PCR plates, tubes and tips should be UV sterilized for 20-30 mins

Publication protocol

The assays were optimized on the Applied Biosystems 7500 FastDX (Life Technologies Corp., NY) instrument, which is widely available in U.S. public health laboratories, and their utility was validated with a large set of clinical samples submitted to the CDC for Rocky Mountain spotted fever testing. The primers for the standard nested PCR assays previously in use at the CDC and the primers, probes, and cycling parameters for the new real-time PCR assays for panrickettsia (PanR8) and R. rickettsii (RRi6) validated in this study. Each 25-μl reaction mixture contained 1× PerfeCTa Multiplex qPCR SuperMix (Quanta, Maryland) with 5 μl DNA template, 1 μM (each) forward and reverse primers, and 40 nM probe concentration; 8 min at 95°C and then 45 cycles of 5 s at 95°C and 30 s at 60°C. The pan-Rickettsia and R. rickettsii real-time PCR assays target the 23S rRNA and a gene encoding hypothetical protein A1G_04230 (GenBank accession no. ABV76353; R. rickettsii Sheila Smith strain), respectively.

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Discussion

Papers

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Manufacturer protocol

Download the product protocol from Quantabio for PerfeCTa MultiPlex qPCR SuperMix below.

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