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Brilliant Core Reagent Kits for QPCR and QRT-PCR

tylcholine receptor g-subunit target. For research parameters that favor convenience, speed, high-throughput capabilities, and reduced risk of sample contamination, the QRT-PCR single-step format is the method of choice. When the highest sensitivity is required, the Brilliant two-step RT-PCR core format is ideal. The Brilliant core reagent kits can be used with various fluorescent chemistries, with or without the passive reference dye, and with UNG decontamination protocols. Furthermore, they compare favorably with other commercially available kits.

Methods

QPCR: A typical 50-l reaction contained the following reagents: 1X Brilliant Plus QPCR core buffer; the optimal concentration of MgCl2 (3.0 to 5.5 mM); sequence-specific primers (300 to 600 nM); probe (200 to 400 nM molecular beacon, 200 to 400 nM TaqMan probe, and 400 nM Scorpions probe); 200 M each dGTP, dATP, dCTP, and 400 M dUTP; 0.5 units of uracil-N-glycosylase ; 2.5 U of SureStart Taq DNA polymerase; and target nucleic acid template (genomic DNA or plasmid DNA as indicated in the Figure legends). Each experiment included control reactions that lacked template-"no-template" controls. All reactions were performed in duplicate. Thermal cycling conditions are as specified for two-step RT-PCR (see below). Other commercially available core reagents or master mixes, TaqMan, and Scorpions chemistries were tested according to the recommendations of each respective manufacturer. The M. tuberculosis rpoB Scorpions and PCR primers are a kind gift of Dr. David Whitcombe of Astra-Zeneca.

Single-Step QRT-PCR: A typical RNA amplification reaction with either molecular beacons or a linear hydrolysis probe contained the following components: 1X Brilliant Plus QRT-PCR buffer 3.0; the optimal concentration of MgCl2 (4.0 to 5.5 mM); 200 M each of dGTP, dATP, dCTP, and 400 M of dUTP; optima
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