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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Rotor-Gene SYBR Green PCR Kit (400)
For 400 x 25 µl reactions: 3 x 1.7 ml 2x Rotor-Gene SYBR Green PCR Master Mix, 2 x 2 ml RNase-Free Water
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204074
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Rotor-Gene SYBR® Green PCR Kit适用于分子生物学应用。该产品不适用于疾病的诊断、预防或治疗。
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).
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BAX (BCL2-associated X protein)
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24.84
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0.05
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29.57
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0.46
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BCL2 (apoptosis gene)
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26.96
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0.05
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32.83
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0.29
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MYC (proto-oncogene)
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28.42
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0.21
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35.26
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0.72
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b-Actin (housekeeping gene)
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20.24
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0.03
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24.39
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0.12
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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").
| 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 |
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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特点
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参数
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应用
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Real-Time quantification of genomic DNA or cDNA targets
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描述
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For ultrafast quantitative real-time PCR and two-step RT-PCR using SYBR Green I on Rotor-Gene cyclers
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反应类型
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Real-time PCR and two-step RT-PCR
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real-time或终点法PCR
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Real-time
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样本/目标类型
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DNA, cDNA
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单一或多重
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Single
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SYBR Green I或序列特异性探针
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SYBR Green I
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热循环仪
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Rotor-Gene Q, Rotor-Gene 3000, Rotor-Gene 6000
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有/无ROX
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Without ROX dye
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For fast real-time PCR, two-step RT-PCR, and one-step RT-PCR using SYBR Green I on Rotor-Gene cyclers
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For genomewide, ready-to-use real-time RT-PCR assays using SYBR Green detection
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图片
高灵敏度、高特异性的检测。
使用10倍稀释的人白细胞cDNA（10 ng到0.1 ng）和QuantiTect Primer Assay对BCL2（CLL/2型淋巴瘤B细胞）进行real-time两步法RT-PCR。反应使用[A] Rotor-Gene SYBR® Green PCR Kit和Rotor-Gene Q实时荧光定量PCR分析仪或[B]供应商AII提供的试剂盒和循环仪进行，重复三次实验。Rotor-Gene体系能够检测到较低的CT值并且有更好的重复性。熔解曲线分析只有一个单峰，表明靶向扩增的特异性。
可靠、特异性的检测，可检测低达10个拷贝。
使用10倍稀释的质粒DNA（109稀释到10个拷贝）和QuantiTect Primer Assay对PPIA（亲环素A）进行real-time PCR。反应使用Rotor-Gene SYBR® Green PCR Kit和Rotor-Gene Q实时荧光定量PCR分析仪，反应重复三次。[A] Rotor-Gene体系在整个浓度范围内检测可靠且CT值重复性好。[B]熔解曲线分析表明靶向扩增的特异性。
Specific primer annealing.
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.
Fast primer annealing.
[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.
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