For real-time PCR and two-step RT-PCR using SYBR Green

• High PCR specificity with integrated hot start
• Reliable quantification of low-abundance transcripts
• Accurate quantification over several logs of template
• Available with or without uracil-N-glycosylase (UNG)
• No need to optimize reaction and cycling conditions

The QuantiTect SYBR Green PCR Kit provides highly specific quantification of gDNA and cDNA targets by real-time PCR and two-step RT-PCR using SYBR Green I detection. The combination of a hot start and a unique qPCR buffer system ensures highly specific and sensitive real-time quantification of gDNA and cDNA targets. The dNTP mix includes dUTP, allowing optional treatment with UNG. For convenience, the master mix can be stored at 2–8°C.

Performance

QuantiTect SYBR Green PCR Kits allow specific quantification over a wide linear range (see figure “Specific quantification over a wide linear range“). HotStarTaq DNA Polymerase provides the most stringent hot start compared with other polymerases, further increasing the specificity of the reaction (see figure “Highly specific amplification“). The unique composition of PCR buffer and HotStarTaq DNA Polymerase enable quantification of even low-abundance transcripts — as few as 5 copies of a target can be accurately detected (see figure “Highly sensitive quantification“). When used in combination with the QuantiTect Reverse Transcription Kit and QuantiTect Primer Assays, the QuantiTect SYBR Green PCR Kit delivers sensitive and reliable results (see figure “Specific and sensitive quantification“).

The combination of the specially optimized UNG solution and the proven PCR master mix in the QuantiTect SYBR Green PCR +UNG Kit ensures effective elimination of carried-over PCR products together with reliable quantification of target sequences (see figure “High specificity in real-time PCR“).

Principle

QuantiTect SYBR Green PCR Kits contain an optimized, ready-to-use master mix for highly specific and sensitive real-time quantification cDNA targets using SYBR Green I. The fluorescent dye SYBR Green I in the master mix enables the analysis of many different targets without having to synthesize target-specific labeled probes. A balanced combination of K⁺ and NH₄⁺ ions in the PCR buffer promotes specific primer annealing and enables high PCR specificity and sensitivity (see figure “Specific primer annealing“). In addition, HotStarTaq DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products.

Applications

The QuantiTect SYBR Green PCR Kit is for use in gene expression analysis of cDNA targets and quantitative gDNA analysis. QuantiTect SYBR Green PCR Kits are compatible with all available real-time cyclers, including instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Agilent. For the Rotor-Gene Q and other Rotor-Gene cyclers, we recommend using the Rotor-Gene SYBR Green PCR Kit, which has been specially developed for fast cycling on these instruments.