QuantiTect SYBR® Green PCR Kits
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.
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List price:
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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
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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
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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
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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
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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.
两步法RT-PCR。|高度特异性扩增。|在ABI PRISM 7700上实现高度灵敏的定量检测。|特异性定量检测。|特异性的灵敏检测。|高度特异性的real-time PCR。|退火时引物特异性结合。|
QuantiTect Probe PCR Kit避免了繁琐且耗时的反应条件优化过程。只需将引物、探针和cDNA模板加入即用型PCR预混液,在任意一台real-time循环仪上开始反应即可。|相比其他DNA聚合酶,只有HotStarTaq DNA Polymerase结合独特的缓冲液可以特异性扩增497 bp的片段(在1 µg人基因组DNA背景下的50个拷贝的HIV-pol基因重组体)。M:分子量标准。|在ABI PRISM 7700上扩增1 x 106至5个拷贝的含有人CFTR基因片段的质粒模板(背景为不含CFTR基因的50 ng玉米基因组DNA)。CFTR(囊性纤维化跨膜传导调节因子)是cAMP调控的氯离子通道。NTC:无模板对照。|在Rotor-Gene 3000上使用针对人BAX的QuantiTect Primer Assay和指定的试剂盒分析稀释的人白细胞cDNA。熔解曲线分析中出现单峰,NTC反应呈平直曲线,说明使用QuantiTect Kit和Assay可实现高特异性定量。NTC:无模板对照。Norm. Fluoro.:标准化的荧光。|在Applied Biosystems 7500上使用针对人IL8的QuantiTect Primer Assay和指定的试剂盒,分析稀释的人白细胞cDNA。熔解曲线分析中出现单峰,NTC反应呈平直曲线,说明只有使用QuantiTect Kit和Assay的组合才可实现高特异性定量。NTC:无模板对照。|使用 [A] QuantiTect SYBR® Green PCR +UNG Kit或 [B] Supplier R的试剂盒和UNG进行UNG预处理的基于SYBR® Green的real-time PCR。在LightCycler 480上使用10倍稀释的人白细胞cDNA(100 ng至100 pg)和针对Myc(一种原癌基因)的QuantiTect Primer Assay运行两次重复反应。熔解曲线分析(参见插图)显示,使用QuantiTect Kit可获得较Supplier R的试剂盒更高的PCR特异性。|退火时,缓冲液中浓度平衡的KCl和(NH4)2SO4促进引物和探针特异性与模板结合。K+结合到双链DNA上的磷酸基团,稳定引物和探针。NH4+则破坏弱的错配碱基之间的氢键。|
性能
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").
原理
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.
QuantiTect SYBR Green PCR master mix also contains dUTP, enabling pretreatment with uracil-N-glycosylase (UNG) prior to starting PCR, which ensures that any contaminating PCR products do not affect subsequent PCR reactions.
| HotStarTaq DNA Polymerase |
15 min activation at 95ºC |
Set-up of qPCR reactions at room temperature |
| QuantiTect SYBR Green PCR Buffer |
Balanced combination of NH4+ and K+ ions |
Specific primer annealing ensures reliable PCR results |
| dNTP mix |
Includes dUTP, which partially replaces dTTP and enables optional UNG treatment of reactions |
Eliminates contamination from carryover of PCR products by optional UNG treatment |
| SYBR Green I dye |
Yields a strong fluorescent signal upon binding double-stranded DNA |
Highly sensitive quantification |
| ROX dye |
For normalization of fluorescent signals on Applied Biosystems and, optionally, Agilent instruments |
Precise quantification on cyclers that require ROX dye. Does not interfere with reactions on other real-time cyclers |
操作流程
QuantiTect SYBR Green PCR Kits overcome the need for optimization of reaction conditions, which can be tedious and time-consuming. Simply add primers and DNA template to the ready-to-use PCR master mix, and start the reaction (see flowchart "Two-step RT-PCR"). Follow the protocol in the handbook to get fast and reliable results on any real-time cycler. If required, reactions can be pretreated with uracil-N-glycosylase (UNG) to eliminate carryover of PCR products from previous reactions.
For optimal results in real-time two-step RT-PCR, we recommend synthesizing cDNA using the QuantiTect Reverse Transcription Kit. The kit provides fast cDNA synthesis in just 20 minutes with integrated removal of genomic DNA contamination.
Highly specific results in gene expression analysis are guaranteed when QuantiTect SYBR Green PCR Kits are used in combination with QuantiTect Primer Assays. These are genomewide, bioinformatically validated primer sets for detecting transcripts from human, mouse, rat, and many other species. QuantiTect Primer Assays can be easily ordered online at GeneGlobe.
应用
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.
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Feature
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Specifications
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Applications
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Real-time quantification of genomic DNA or cDNA targets
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Reaction type
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Real-time PCR and two-step RT-PCR
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Real-time or endpoint
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Real-time
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Sample/target type
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DNA, cDNA
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Single or multiplex
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Single
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SYBR Green I or sequence-specific probes
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SYBR Green I
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Thermal cycler
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All real-time cyclers (e.g. LC, RG, ABI)
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With or without ROX
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With ROX
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For use with QuantiTect PCR Kits to eliminate carryover of PCR products
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Show details
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For genomewide, ready-to-use real-time RT-PCR assays using SYBR Green detection
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Show details
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For quantitative, real-time PCR and two-step RT-PCR using SYBR Green I
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Show details
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Download Safety Data Sheets for QIAGEN product components.
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Images
Two-step RT-PCR.
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.
Highly specific amplification.
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.
Highly sensitive quantification on the ABI PRISM 7700.
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.
Specific quantification over a wide linear range.
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.
Specific and sensitive quantification.
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.
High specificity in real-time PCR.
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.
Specific primer annealing.
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.
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