Rotor-Gene SYBR® Green PCR Kit

For ultrafast, real-time PCR and two step RT-PCR using SYBR Green on Rotor-Gene cyclers

  • Specific and sensitive detection of even low-copy targets
  • Accurate detection of a wide range of template amounts
  • Specially formulated master mix for reliable results without optimization
  • Guaranteed performance combined with QuantiTect Primer Assays

The Rotor-Gene SYBR Green PCR Kit is designed for use with the Rotor-Gene Q and other Rotor-Gene cyclers, providing ultrafast, highly specific quantification of gDNA and cDNA targets with quantitative real-time PCR or two-step RT-PCR using SYBR Green I detection. Outstanding qPCR performance is achieved through the combination of a specially optimized master mix and the unique Rotor-Gene cycler. For convenience, the master mix can be stored at 2–8°C.

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The Rotor-Gene SYBR® Green PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Highly specific and sensitive detection.|Reliable and specific detection down to 10 copies.|Specific primer annealing.|Fast primer annealing.|
Real-time two-step RT-PCR was carried out using tenfold dilutions of human leukocyte cDNA (10 ng to 0.1 ng) and a QuantiTect Primer Assay for BCL2 (B-cell CLL/lymphoma 2). Reactions were run in triplicate using either [A] the Rotor-Gene SYBR® Green PCR Kit and Rotor-Gene Q or [B] a kit and cycler from Supplier AII. The Rotor-Gene system provided lower CT values and greater reproducibility within triplicates. Melting curve analysis (see insets) showed a single peak, demonstrating specific amplification of the target.|Real-time PCR was carried out using tenfold dilutions of plasmid DNA (109 to 10 copies) and a QuantiTect Primer Assay for PPIA (cyclophilin A). Reactions were run in triplicate using the Rotor-Gene SYBR® Green PCR Kit and Rotor-Gene Q. [A] The Rotor-Gene system provided reliable detection over the entire range of template dilutions as well as highly reproducible CT values within each set of triplicates. [B] Melting curve analysis indicates specific amplification of the target.|Cations in the Rotor-Gene Q PCR buffer increase specific primer annealing. K+ binds to phosphate groups on double-stranded DNA, stabilizing primer annealing. NH4+ destabilizes weak hydrogen bonds between mismatched bases.|[A] Q-Bond in Rotor-Gene SYBR® Green PCR Master Mix increases the affinity of DNA polymerase for short single-stranded DNA, reducing primer annealing time to a few seconds. In addition, the unique buffer composition supports the melting of DNA, reducing denaturation and extension times. [B] Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing primer annealing time.|
Performance

The Rotor-Gene SYBR Green PCR Kit is well suited for use in gene expression analysis of cDNA targets (see figures "Highly specific and sensitive detection" and "Reliable and specific detection down to 10 copies").

When QuantiTect Primer Assays are used together with the Rotor-Gene SYBR Green PCR Kit, highly sensitive quantification of specific PCR products is achieved without the need for optimization (see table).

Superior performance in RT-PCR with SYBR Green
QIAGEN Supplier AII
CT Mean deviation CT Mean deviation
BAX (BCL2-associated X protein) 24.84 0.05 29.57 0.46
BCL2 (apoptosis gene) 26.96 0.05 32.83 0.29
MYC (proto-oncogene) 28.42 0.21 35.26 0.72
b-Actin (housekeeping gene) 20.24 0.03 24.39 0.12
Human leukocyte cDNA (1 ng) was used as a template in SYBR Green-based real-time two-step RT-PCR. Triplicate reactions were run using QuantiTect Primer Assays for 4 different targets: BAX, BCL2, MYC, and β-Actin.
Principle

The Rotor-Gene SYBR Green PCR Kit enables rapid and reliable real-time PCR quantification on the Rotor-Gene Q without the need for optimization of reaction and cycling conditions. 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. Highly specific amplification is assured through a balanced combination of K+ and NH4+ ions, which promote specific primer annealing, enabling high PCR specificity and sensitivity (see figure "Specific primer annealing"). Fast cycling without compromising performance is achieved using Q-Bond, a novel PCR additive that enables cycler run times of as low as 45 minutes (see figure "Fast primer annealing").

Components of 2x Rotor-Gene SYBR Green PCR Kit*
ComponentFeatures Benefits
HotStarTaq Plus DNA Polymerase 5 min activation at 95ºC Set up of qPCR reactions at room temperature
Rotor-Gene SYBR Green PCR Buffer Balanced combination of NH4+ and K+ ions Specific primer annealing ensures reliable qPCR results
Unique Q-Bond additive Faster PCR run times enable faster results and more reactions per day
SYBR Green I dye Yields a strong fluorescent signal upon binding double-stranded DNA Highly sensitive quantification
* Also contains dNTP mix (dATP, dCTP, dGTP, dTTP).
Procedure

The Rotor-Gene SYBR Green PCR Master Mix eliminates the need for optimization of reaction and cycling conditions. Simply add DNA template DNA and primers to the master mix and program the cycler. Instructions are provided in the detailed handbook supplied with the kit.

For gene expression analysis using real-time two-step RT-PCR, the combination of the Rotor-Gene SYBR Green PCR Kit, QuantiTect Reverse Transcription Kit, QuantiTect Primer Assays, and Rotor-Gene Q provides a complete, ready-to-run solution. The QuantiTect Reverse Transcription Kit delivers fast cDNA synthesis in just 20 minutes with integrated removal of genomic DNA contamination. QuantiTect Primer Assays are bioinformatically validated primer sets for any gene from human, mouse, rat, and many other species. Assays can be easily ordered online at the GeneGlobe Web portal.

Applications

The Rotor-Gene SYBR Green PCR Kit provides rapid real-time quantification of cDNA  and gDNA targets on the Rotor-Gene Q. The Kits are also compatible with the Rotor-Gene 3000 and the Rotor-Gene 6000.

For ultrafast, one-step qRT-PCR gene expression analysis of RNA targets using SYBR Green I on Rotor-Gene cyclers, use the Rotor-Gene SYBR Green RT-PCR Kit.

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Feature
Specifications
Applications Real-Time quantification of genomic DNA or cDNA targets
Description For ultrafast quantitative real-time PCR and two-step RT-PCR using SYBR Green I on Rotor-Gene cyclers
Reaction type Real-time PCR and two-step RT-PCR
Real-time or endpoint Real-time
Sample/target type DNA, cDNA
Single or multiplex Single
SYBR Green I or sequence-specific probes SYBR Green I
Thermal cycler Rotor-Gene Q, Rotor-Gene 3000, Rotor-Gene 6000
With or without ROX Without ROX dye
Sample to Insight
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