QuantiTect SYBR® Green PCR Kits

使用SYBR Green I进行两步法RT-PCR,用于基因表达分析

  • 整合热启动,PCR特异性高
  • 可靠定量检测低丰度转录本
  • 准确定量检测各种起始量样本
  • 提供含有或不含尿嘧啶DNA糖基化酶(UNG)两种包装
  • 无需优化反应和循环条件

QuantiTect SYBR Green PCR Kit使用SYBR Green I通过两步法real-time RT-PCR对cDNA目标序列进行高特异性的定量检测。热启动和独特的实时定量PCR缓冲液确保了对cDNA目标序列高特异性和灵敏度的real-time定量检测。dNTP混合液含有dUTP,可选择性进行UNG处理。为便于使用,预混液可储存在2–8°C。

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QuantiTect SYBR Green PCR Kit (40)
Trial kit for 40 x 50 µl reactions: 1 ml 2x QuantiTect SYBR Green PCR Master Mix, 2 ml RNase-Free Water
204141
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QuantiTect SYBR Green PCR Kit (200)
For 200 x 50 µl reactions: 3 x 1.7 ml 2x QuantiTect SYBR Green PCR Master Mix, 2 x 2 ml RNase-Free Water
204143
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QuantiTect SYBR Green PCR Kit (1000)
For 1000 x 50 µl reactions: 25 ml 2x QuantiTect SYBR Green PCR Master Mix, 20 ml RNase-Free Water
204145
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QuantiTect SYBR Green PCR +UNG Kit (200)
For 200 x 50 µl reactions: 3 x 1.7 ml 2x QuantiTect SYBR Green PCR Master Mix, 100 µl UNG Solution, 2 x 2 ml RNase-Free Water
204163
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QuantiTect SYBR® Green PCR Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.


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Two-step RT-PCR.|Highly specific amplification.|Highly sensitive quantification on the ABI PRISM 7700.|Specific quantification over a wide linear range.|Specific and sensitive quantification.|High specificity in real-time PCR.|Specific primer annealing.|
The QuantiTect SYBR® Green PCR Kit overcomes the need for optimization of reaction conditions, which can be tedious and time-consuming. Simply add primers and template to the ready-to-use PCR master mix, and start the reaction on any real-time cycler.|In comparison with other DNA polymerases, only HotStarTaq DNA Polymerase in combination with a unique buffer specifically amplified a 497 bp fragment (from 50 copies of an HIV-pol-gene construct in a background of 1 µg human genomic DNA). M: Markers.|Template ranging from 1 x 106 to 5 copies of a plasmid containing a human CFTR gene fragment (in a background of 50 ng maize genomic DNA with no CFTR gene) was amplified on the ABI PRISM 7700. CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-regulated chloride channel. NTC: No template control.|Dilutions of human leukocyte cDNA were analyzed using the QuantiTect Primer Assay for human BAX and the indicated kits on the Rotor-Gene 3000. Highly specific quantification with the QuantiTect Kit and Assay is demonstrated by the single peak in melting curve analysis and the flat curve for the NTC reaction. NTC: No template control. Norm. Fluoro.: Normalized fluorescence.|Dilutions of human leukocyte cDNA were analyzed using the QuantiTect Primer Assay for human IL8 and the indicated kits on the Applied Biosystems 7500. Only the combination of QuantiTect Kit and Assay provided highly specific quantification, as demonstrated by the single peak in melting curve analysis and the flat curve for the NTC reaction. NTC: No template control.|SYBR® Green-based real-time PCR with UNG pretreatment was carried out using either [A] the QuantiTect SYBR® Green PCR +UNG Kit or [B] a kit and UNG from Supplier R. Reactions were run in duplicate on the LightCycler 480 using 10-fold dilutions of human leukocyte cDNA (100 ng to 100 pg) and a QuantiTect Primer Assay for Myc (a protooncogene). Melting curve analysis (see insets) revealed higher PCR specificity with the QuantiTect Kit than with the kit from Supplier R.|A balanced combination of KCl and (NH4)2SO4 promotes specific annealing of primers to the PCR template. K+ binds to phosphate groups on double-stranded DNA, stabilizing primer annealing. NH4+ destabilizes weak hydrogen bonds between mismatched bases.|
Performance

QuantiTect SYBR Green PCR Kit在宽泛的范围内符合线性规律,可实现特异性定量检测(参见"Specific quantification over a wide linear range")。与其他聚合酶相比,HotStarTaq DNA Polymerase具有严格的热启动,进一步提高了PCR反应的特异性。独特的PCR缓冲液和HotStarTaq DNA Polymerase,确保低丰度转录本的定量检测,低至5个拷贝的目标序列也可被准确检测(参见"Highly specific amplification")。QuantiTect SYBR Green PCR Kit与QuantiTect Reverse Transcription Kit和QuantiTect Primer Assays配合使用,可获得灵敏、可靠的结果(参见"Specific and sensitive quantification")。

QuantiTect SYBR Green PCR +UNG Kit中经特别优化的UNG溶液配合经验证的PCR预混液可有效避免PCR产物中的残留污染,并对靶序列进行可靠的定量检测(参见"High specificity in real-time PCR")。

Principle

QuantiTect SYBR Green PCR Kit含有优化的即用型预混液,可使用SYBR Green I对cDNA靶序列进行高特异性和灵敏度的real-time定量分析。预混液中的荧光染料SYBR Green I可对很多不同的靶序列进行分析,无需合成靶标特异性标记引物。独特的PCR缓冲液含有K+和NH4+,能促使特异性引物退火,获得高PCR特异性和灵敏度(参见"Specific primer annealing")。此外,HotStarTaq DNA Polymerase具有严格的热启动,可防止非特异性产物的形成。

QuantiTect SYBR Green PCR预混液含有dUTP,可在PCR前使用尿嘧啶DNA糖基化酶 (UNG)预处理,保证之前反应残留的PCR产物会影响后续的PCR反应。

2x QuantiTect SYBR Green PCR Kit的组份
组份 特点 优势
HotStarTaq DNA Polymerase 95ºC孵育15分钟即可活化 室温下即可构建qPCR反应体系
QuantiTect SYBR Green PCR Buffer NH4+和K+的平衡组合 特异性引物退火确保PCR结果可靠
dNTP mix 含有dUTP,会部分替代dTTP,可进行UNG处理 可选择进行UNG处理,以消除之前反应残留的PCR产物
SYBR Green I dye DNA双链结合时,生成强烈的荧光信号 高度灵敏的定量检测
ROX dye 在Applied Biosystems或Agilent的PCR仪上进行荧光信号校准 可在需要ROX染料的PCR仪上进行准确的定量检测。不会干扰其他real-time PCR仪
Procedure

QuantiTect SYBR Green PCR Kit无需进行繁琐耗时的反应条件优化。只需简单地将引物、探针和cDNA模板加入到即用型PCR预混液中,即可开始反应(参见"Two-step RT-PCR")。使用操作手册中的实验方案可在任何real-time PCR仪上得到快速、可靠的结果。如果需要,反应液可用尿嘧啶DNA糖基化酶(UNG)预处理,以去除之前反应残留的PCR产物。

为在两步法real-time RT-PCR中获得理想结果,我们推荐使用QuantiTect Reverse Transcription Kit合成cDNA。此试剂盒可在20分钟内快速合成cDNA,并可去除基因组DNA污染。

QuantiTect SYBR Green PCR Kit配合QuantiTect Primer Assays可在基因表达分析中获得高度特异性的结果。基因组范围、生物信息学验证的引物对覆盖所有人类、小鼠、大鼠和其他各类生物基因。QuantiTect Primer Assays可在GeneGlobe网站上方便地订购。

Applications

QuantiTect SYBR Green PCR Kit可用在各种real-time PCR仪上对cDNA目标序列进行基因表达分析,包括Applied Biosystems、Bio-Rad、Cepheid、Eppendorf、Roche和Agilent的仪器。在Rotor-Gene Q实时荧光定量PCR分析仪上使用时,我们推荐专门为快速PCR研发的Rotor-Gene SYBR Green PCR Kit。

Feature
Specifications
Applications Real-time quantification of genomic DNA or cDNA targets
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 All real-time cyclers (e.g. LC, RG, ABI)
With or without ROX With ROX

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Kit Handbooks
3
For use with QuantiTect PCR Kits to eliminate carryover of PCR products
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For genomewide, ready-to-use real-time RT-PCR assays using SYBR Green detection
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For quantitative, real-time PCR and two-step RT-PCR using SYBR Green I
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