FAQ-1146
Can I increase the annealing temperature recommended for QuantiTect Primer Assays used with the QuantiTect SYBR Green PCR Kits?
QuantiTect Primer Assays are optimized for an annealing temperature of 55ºC when using the QuantiTect SYBR Green PCR Kit (for two-step RT-PCR) or the QuantiTect SYBR Green RT-PCR Kit (for one-step RT-PCR) .
Increasing the annealing temperature will reduce PCR performance (sensitivity and PCR efficiency) when using QuantiTect Primer Assays with these kits.
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QuantiTect Primer Assays
QuantiTect Primer Assays are genomewide, bioinformatically validated primer sets for use in SYBR Green-based real-time RT-PCR on any cycler. Assays are available for all genes from human, rat, mouse, and many other species. Each assay for a specific gene is supplied as a lyophilized mix of forward and reverse primers that can be easily reconstituted to obtain a 10x assay solution (reaction components for real-time RT-PCR need to be ordered separately). When used in combination with QuantiFast, QuantiTect, Rotor-Gene, or FastLane Kits for SYBR Green detection, QuantiTect Primer Assays guarantee highly specific and sensitive results in real-time RT-PCR that are comparable to probe-based detection.
QuantiTect RT-PCR Kits
QuantiTect RT-PCR Kits enable sensitive quantification of RNA targets by real-time one-step PCR using sequence-specific probes or SYBR Green I detection. The kits also allow reliable quantification of up to 5 RNA targets in a single tube by multiplex, real-time one-step RT-PCR. The combination of a hot start and a unique PCR buffer system in the ready-to-use master mix ensures highly sensitive qRT-PCR on any real-time cycler without the need for optimization. The dNTP mix includes dUTP, allowing optional treatment with UNG. For convenience, the master mixes in QuantiTect one-step RT-PCR kits can be stored at 2–8°C. Two kit formats are available for multiplex RT-PCR using sequence-specific probes: the QuantiTect Multiplex RT-PCR Kit for cyclers that require ROX dye for fluorescence normalization, and the QuantiTect Multiplex RT-PCR NoROX Kit for all other cyclers. The QuantiTect SYBR Green RT-PCR Kit is supplied with an optimized RT mix for efficient and sensitive reverse transcription over a wide range of RNA template amounts.
QuantiTect PCR Kits
QuantiTect PCR Kits (qPCR kits) enable sensitive quantification of gDNA and cDNA targets by real-time PCR and two-step RT-PCR using sequence-specific probes or SYBR Green I detection. The real-time PCR kits also allow reliable quantification of up to 5 gDNA or cDNA targets in a single tube by multiplex, real-time PCR or two-step RT-PCR. The combination of a hot start and a unique PCR buffer system in the ready-to-use master mix ensures highly sensitive qPCR on any real-time cycler without the need for optimization. The dNTP mix includes dUTP, allowing optional treatment with UNG. For convenience, the master mixes in QuantiTect PCR Kits can be stored at 2–8°C. Two kit formats are available for multiplex PCR using sequence-specific probes: the QuantiTect Multiplex PCR Kit for cyclers that require ROX dye for fluorescence normalization, and the QuantiTect Multiplex PCR NoROX Kit for all other cyclers.