QuantiTect Probe PCR Kits

For real-time PCR and two-step RT-PCR using sequence-specific probes

  • Highly sensitive detection of low-copy targets
  • Accurate quantification over several logs of template
  • Use of any sequence-specific probe on any real-time cycler
  • Available with or without uracil-N-glycosylase (UNG)
  • No need to optimize reaction and cycling conditions

QuantiTect Probe PCR Kits enable sensitive quantification of gDNA and cDNA targets by real-time PCR and two-step RT-PCR using sequence-specific probes. The combination of a hot start and a unique PCR buffer system in the ready-to-use master mix ensures highly sensitive qPCR on any real-time cycler without the need for optimization. The dNTP mix includes dUTP, allowing optional treatment with UNG. For convenience, the master mix can be stored at 2–8°C.

产品 货号 目录价:
QuantiTect Probe PCR Kit (200)
For 200 x 50 µl reactions: 3 x 1.7 ml 2x QuantiTect Probe PCR Master Mix, 2 x 2 ml RNase-Free Water
204343
询价
QuantiTect Probe PCR Kit (1000)
For 1000 x 50 µl reactions: 25 ml 2x QuantiTect Probe PCR Master Mix, 20 ml RNase-Free Water
204345
询价
QuantiTect Probe PCR +UNG Kit (200)
For 200 x 50 µl reactions: 3 x 1.7 ml 2x QuantiTect Probe PCR Master Mix, 100 ul UNG Solution, 2 x 2 ml RNase-Free Water
204363
询价

QuantiTect Probe PCR Kits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。


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高效的UNG酶切。|两步法RT-PCR。|高度特异性扩增。|两步法PCR实现对低至高模板量的准确分析。|real-time PCR实现对低至高模板量的准确检测。|高灵敏度、高效率。|退火时引物特异性结合。|
采用UNG处理或不处理两个106个拷贝的含有dUMP的PCR扩增片段,然后进行real-time PCR扩增。UNG来自不同的供应商,使用QuantiTect Probe PCR +UNG Kit中的预混液进行real-time PCR。Y轴上的ΔCT表示UNG处理的样本的CT值减去未经UNG处理的样本的CT值。QuantiTect Kit提供的UNG具有最高的ΔCT (9-12个循环)。这说明QIAGEN UNG对残留PCR产物的酶切效率高于其他供应商的UNG。|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Mx3005P上使用10倍稀释的人白细胞cDNA (10 ng至0.01 ng) 和针对IL1R2 (一种细胞因子) 的TaqMan Assay进行两次重复反应。使用QuantiTect Probe PCR Kit可实现从低模板量至高模板量的准确基因表达分析,PCR效率为101%。|使用QuantiTect Probe PCR +UNG Kit进行UNG预处理的基于探针的real-time PCR。在ABI PRISM 7900上使用10倍稀释的人白细胞cDNA (100 ng至10 pg) 和针对IL8 (一种细胞因子) 的TaqMan Assay进行两次重复反应。扩增曲线分布均匀,PCR效率高达94%。|在Mx3005上使用QuantiTect Probe PCR Kit及IL8 (白介素8) 特异性的引物和FAM标记的探针分析10倍连续稀释的白细胞cDNA (100 ng至1 pg),反应重复两次。在6 log模板稀释度范围内的PCR效率达97%,可实现低至1 pg的IL8转录本的灵敏检测。|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

The QuantiTect Probe PCR Kit delivers sensitive and reliable results in combination with the QuantiTect Reverse Transcription Kit (see figure "High sensitivity and efficiency, and wide dynamic range"). The unique PCR buffer composition enables QuantiTect Probe PCR Kits to provide sensitive quantification of low-copy DNA targets, as well as accurate quantification over a wide linear range (see figures "High sensitivity and efficiency, and wide dynamic range" and "Wide dynamic range in two-step RT-PCR"). HotStarTaq DNA Polymerase further increases the specificity of the PCR reaction by providing the most stringent hot start compared with other polymerases (see figure "Highly specific amplification").

A major source of PCR contamination is the carryover of PCR products from previous reactions. Pretreatment with UNG prior to starting PCR ensures that any contaminating PCR products do not affect subsequent PCR. The combination of the specially optimized UNG solution and the proven PCR master mix in the QuantiTect Probe PCR +UNG Kit ensures effective elimination of carried-over PCR products together with reliable quantification of target sequences (see figures "Effective UNG digestion" and "Wide dynamic range in real-time PCR").

原理

QuantiTect Probe PCR Kits contain an optimized, ready-to-use master mix for highly specific and sensitive real-time quantification of gDNA and cDNA targets using sequence-specific probes. The kits are designed for use with all types of sequence-specific probes, including hydrolysis probe detection (e.g., TaqMan® and other dual-labeled probes), FRET probes, and Molecular Beacons. QuantiTect Probe PCR Kits contain a unique PCR buffer that contains a balanced combination of K+ and NH4+ ions, which promote specific primer annealing, enabling 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 Probe 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.

Components of 2x QuantiTect Probe PCR Kit
Component Features Benefits
HotStarTaq DNA Polymerase 15 min activation at 95ºC Set-up of qPCR reactions at room temperature
QuantiTect Probe 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
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 Probe PCR Kits overcome the need for optimization of reaction conditions, which can be tedious and time-consuming. Simply add primers, probe, 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.

应用

QuantiTect Probe PCR Kits can be used for probe-based gene expression analysis of cDNA targets or quantitative gDNA analysis on any real-time cycler. This includes 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 Probe PCR Kit, which has been specially developed for fast cycling on these instruments.

特点
参数
应用 Real-time quantification of DNA, cDNA, or RNA targets
反应类型 PCR and two-step RT-PCR
real-time或终点法PCR Real-time
样本/目标类型 DNA, cDNA, RNA
单一或多重 Single
SYBR Green I或序列特异性探针 Sequence-specific probes
热循环仪 Most real-time cyclers (e.g. LC, RG, ABI)
有/无ROX With ROX

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试剂盒操作手册
2
For use with QuantiTect PCR Kits to eliminate carryover of PCR products
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For quantitative, real-time PCR and two-step RT-PCR using sequence-specific probes
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安全数据表
1
Download Safety Data Sheets for QIAGEN product components.
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图片
Effective UNG digestion.
高效的UNG酶切。
采用UNG处理或不处理两个106个拷贝的含有dUMP的PCR扩增片段,然后进行real-time PCR扩增。UNG来自不同的供应商,使用QuantiTect Probe PCR +UNG Kit中的预混液进行real-time PCR。Y轴上的ΔCT表示UNG处理的样本的CT值减去未经UNG处理的样本的CT值。QuantiTect Kit提供的UNG具有最高的ΔCT (9-12个循环)。这说明QIAGEN UNG对残留PCR产物的酶切效率高于其他供应商的UNG。
PCR and two-step RT-PCR.
两步法RT-PCR。
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。
Highly specific amplification.
高度特异性扩增。
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。
Wide dynamic range in two-step RT-PCR
两步法PCR实现对低至高模板量的准确分析。
在Mx3005P上使用10倍稀释的人白细胞cDNA (10 ng至0.01 ng) 和针对IL1R2 (一种细胞因子) 的TaqMan Assay进行两次重复反应。使用QuantiTect Probe PCR Kit可实现从低模板量至高模板量的准确基因表达分析,PCR效率为101%。
Wide dynamic range in real-time PCR.
real-time PCR实现对低至高模板量的准确检测。
使用QuantiTect Probe PCR +UNG Kit进行UNG预处理的基于探针的real-time PCR。在ABI PRISM 7900上使用10倍稀释的人白细胞cDNA (100 ng至10 pg) 和针对IL8 (一种细胞因子) 的TaqMan Assay进行两次重复反应。扩增曲线分布均匀,PCR效率高达94%。
High sensitivity and efficiency, and wide dynamic range.
高灵敏度、高效率。
在Mx3005上使用QuantiTect Probe PCR Kit及IL8 (白介素8) 特异性的引物和FAM标记的探针分析10倍连续稀释的白细胞cDNA (100 ng至1 pg),反应重复两次。在6 log模板稀释度范围内的PCR效率达97%,可实现低至1 pg的IL8转录本的灵敏检测。
Specific primer annealing using a balanced combination of K+ and NH4+ ions
退火时引物特异性结合。
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。