Rotor-Gene SYBR® Green PCR Kit

在Rotor-Gene PCR仪上使用SYBR Green I通过两步法qRT-PCR超快速进行基因表达分析

  • 特异性检测低拷贝靶基因
  • 精确检测广泛模板量范围内的基因
  • 特制的即用型预混液,用于快速循环
  • 配合QuantiTect Primer Assays使用,可保证性能

Rotor-Gene SYBR Green PCR Kit专用于Rotor-Gene Q实时荧光定量PCR分析仪和其他的Rotor-Gene PCR仪,使用SYBR Green I对cDNA靶基因进行超快速、高特异性的real-time PCR和两步法RT-PCR分析。经优化的预混液与独特的Rotor-Gene PCR仪配合使用保证了qPCR良好的性能。预混液可贮存在2–8°C,方便使用。

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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
204074
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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

Rotor-Gene SYBR Green PCR Kit特别适用于对cDNA靶基因进行基因表达分析(参见"Highly specific and sensitive detection"和"Reliable and specific detection down to 10 copies")。

QuantiTect Primer Assays与Rotor-Gene SYBR Green PCR Kit配合使用,可高灵敏度定量检测特异性PCR产物,无需经过优化(参见下表)。

使用SYBR Green在RT-PCR中获得优越性能
QIAGEN Supplier AII
CT 平均偏差 CT 平均偏差
BAX (BCL2-associated X protein) 24.84 0.05 29.57 0.46
BCL2 (凋亡基因) 26.96

0.05

32.83 0.29
MYC (原癌细胞) 28.42 0.21 35.26 0.72
b-Actin (管家基因) 20.24 0.03 24.39 0.12
SYBR Green-based real-time两步法RT-PCR用人类白血球cDNA(1ng)做样本。使用QuantiTect Primer Assays对4种不同的靶基因:BAX、BCL2、MYC和β-Actin进行3次测定
Principle

Rotor-Gene SYBR Green PCR Kit可在Rotor-Gene Q实时荧光定量PCR分析仪上进行快速、可靠的real-time PCR定量分析,无需优化反应及循环条件。混合液中的荧光染料SYBR Green I能用于分析多种不同的靶基因而无需合成靶标特异性标签探针。预混液中浓度平衡的K+和NH4+离子组合保证了高特异性扩增,使引物退火高度特异性,获得高PCR特异性和灵敏性(参见"Specific primer annealing")。使用创新的PCR辅助剂Q-Bond使循环时间降低至45分钟,达到快速分析且不降低性能(参见"Fast primer annealing")。

2x Rotor-Gene SYBR Green PCR Kit的组分*
组分特点 优势
HotStarTaq Plus DNA Polymerase 在95ºC下5分钟活化 室温下进行qPCR反应体系构建
Rotor-Gene SYBR Green PCR Buffer 浓度平衡的K+和NH4+离子组合 引物特异性结合保证可靠的qPCR结果
独特的Q-Bond添加剂 快速的PCR运行时间可获得较快的结果和每天更多的反应次数
SYBR Green I染料 与双链DNA结合产生较强的荧光信号 高度灵敏的定量检测
* 同时含有dNTP预混液(dATP, dCTP, dGTP, dTTP)。
Procedure

Rotor-Gene SYBR Green PCR Master Mix无需优化反应及循环条件。只需将cDNA模板DNA与引物加入到预混液中并设置循环程序。在试剂盒使用手册中有详细说明。

对于基于real-time两步法RT-PCR的基因表达分析,Rotor-Gene SYBR Green PCR Kit、QuantiTect Reverse Transcription Kit、QuantiTect Primer Assays及Rotor-Gene Q实时荧光定量PCR分析仪提供了完成的即用型解决方案。QuantiTect Reverse Transcription Kit可以在20分钟内快速合成cDNA并完全去除基因组DNA污染。QuantiTect Primer Assays为覆盖全基因组,经功效验证的预制引物对,用于检测来自人、小鼠、大鼠和其他物种的转录本。QuantiTect Primer Assays可以便利的在GeneGlobe网上订购。

Applications

Rotor-Gene SYBR Green PCR Kit适用于在Rotor-Gene Q实时荧光定量PCR分析仪上对cDNA靶基因进行快速real-time定量分析。该试剂盒与Rotor-Gene 3000和Rotor-Gene 6000兼容。

可使用Rotor-Gene SYBR Green RT-PCR Kit在Rotor-Gene PCR仪上通过SYBR Green I对目标RNA进行超快速、一步法qRT-PCR基因表达分析。

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

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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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