QuantiFast Probe PCR Kits
For fast, real-time PCR and two-step qRT-PCR using sequence-specific probes
- Sensitive detection of even low copy targets
- Accurate detection of a wide range of template amounts
- Faster results with a time savings of up to 60%
- Optimized, ready-to-use master mix for reliable results without optimization
- One cycling protocol for all standard and fast cyclers
QuantiFast Probe PCR Kits deliver fast and sensitive quantification of gDNA and cDNA targets by real-time PCR and two-step RT-PCR using sequence-specific probes. Q-bond technology and an optimized master mix enable shorter real-time PCR run times, not only on fast cyclers with short ramping times, but also on standard cyclers. 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. Two kit formats are available: the QuantiFast Probe PCR Kit for cyclers that require ROX dye for fluorescence normalization, and the QuantiFast Probe PCR +ROX Vial Kit for all other cyclers. For convenience, the master mix can be stored at 2–8°C.
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QuantiFast Probe PCR Kit (4000)
For 4000 x 25 µl reactions: 2 x 25 ml 2x QuantiFast Probe PCR Master Mix (contains ROX dye), 20 ml RNase-Free Water
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204257
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QuantiFast 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。
See trademarks.
Significantly reduced PCR times.|Wide dynamic range and high sensitivity.|Faster results without compromising sensitivity.|Sensitive two-step RT-PCR.|Detection over 7 Logs of template.|Specific primer annealing.|Fast primer annealing.|
QuantiFast Probe Kits reduce total PCR run time by up to 60% in real-time two-step RT-PCR on standard cyclers (40 PCR cycles carried out; comparison with a standard QIAGEN real-time PCR kit). L: LightCycler 2.0; A1: ABI PRISM 7900; A2: Applied Biosystems 7500; A3: ABI PRISM 7000. |The QuantiFast Probe PCR +ROX Vial Kit provided accurate gene expression analysis from low to high template amounts with a PCR efficiency of 93% in two-step PCR on the Mastercycler ep realplex. Duplicate reactions (25 μl volume) were performed using 10-fold dilutions of human leukocyte cDNA and a Primer Express designed TaqMan assay for ubiquitin (a regulatory protein). NTC: no template control.|Reactions were run in duplicate using 10-fold dilutions of human leukocyte cDNA (100 ng to 0.01 ng) and a TaqMan gene expression assay for IL1RN (a cytokine) on the ABI PRISM 7900. The QuantiFast Kit showed greater sensitivity than the standard cycling kit from Supplier AII (which was used according to the standard cycling protocol), providing much lower CT values and transcript quantification from down to 10 pg cDNA.|Reactions were run in triplicate using 10-fold dilutions of human leukocyte cDNA (100 ng to 0.01 ng) and a Primer Express designed TaqMan assay for IL8 (a chemokine) on the ABI PRISM 7900. [A] The QuantiFast Kit showed greater sensitivity than [B] the kit from Supplier I (which was used according to the standard cycling protocol), providing lower CT values and transcript quantification from down to 0.01 ng cDNA.|Duplicate two-step RT-PCR reactions were run using a set of primers and FRET probes for b-2 microglobulin from Supplier R on the LightCycler 2.0. The amplification plots for the QuantiFast Kit show the kit's wide dynamic range.|A balanced combination of KCl and (NH4)2SO4 promotes specific annealing of primers and probes to the PCR template. K+ binds to phosphate groups on double-stranded DNA, stabilizing annealing of primers and probes. NH4+ destabilizes weak hydrogen bonds between mismatched bases.|[A] Q-Bond in QuantiFast Buffer 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
QuantiFast Probe PCR Kits deliver highly sensitive results, outperforming other real-time PCR kits (see table "Sensitive two-step RT-PCR on the iCycler iQ" and figure and Sensitive two-step RT-PCR). PCR run times are reduced by up to 60% (see figure "Significantly reduced PCR times"), allowing you to achieve fast PCR results without compromising on PCR performance (see figure "Faster results without compromising sensitivity"). You can also greatly increase your sample throughput or efficiently share a cycler with other users.
| 100 |
29.13 |
31.13 |
| 10 |
32.50 |
34.43 |
| 1 |
36.15 |
40.00 |
| No template control |
40.00 |
40.00 |
QuantiFast Probe PCR Kits allow accurate quantification over a wide dynamic range (see figure "Wide dynamic range and high sensitivity"). Template dilutions of up to 7 logs can be reliably detected (see table "Detection over 7 logs of template in two-step RT-PCR on the LightCycler 2.0 " and figure "Detection over 7 logs of template").
| 100 ng |
11.99 |
14.56 |
| 10 ng |
15.54 |
17.99 |
| 1 ng |
19.22 |
22.16 |
| 100 pg |
22.63 |
25.14 |
| 10 pg |
26.43 |
30.15 |
| 1 pg |
30.02 |
34.43 |
| 100 fg |
33.42 |
37.10 |
| No template control |
40 |
40.00 |
| PCR efficiency |
90% |
80.1% |
Principle
QuantiFast Probe PCR Kits deliver highly sensitive and rapid results over a wide dynamic range on both standard and fast cyclers. 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) and FRET probes. A specially developed, fast PCR buffer contains the novel additive Q-Bond, which significantly reduces denaturation, annealing, and extension times (see figure "Fast primer annealing"). The buffer also 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 Plus DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products.
| HotStarTaq Plus DNA Polymerase |
3 min activation at 95ºC |
Set up of qPCR reactions at room temperature |
| QuantiFast Probe PCR Buffer |
Balanced combination of NH4+ and K+ ions |
Specific primer annealing ensures reliable PCR results |
| Unique Q-Bond additive |
Faster PCR run times, enabling faster results and more reactions per day |
| ROX dye† |
Normalizes fluorescent signals on Applied Biosystems and, optionally, Agilent instruments |
Precise quantification on cyclers that require ROX dye. Does not interfere with PCR on any real-time cycler |
Procedure
QuantiFast Probe PCR Kits contain ready-to-use master mixes that eliminate the need for optimization of reaction and cycling conditions. Simply add template DNA, primers, and probe to the master mix and follow the protocol in the handbook to get fast and reliable results on any real-time cycler. Kits are available with or without ROX passive reference dye in the master mix, enabling use on virtually any real-time cycler (see table). Due to the optimized ROX concentrations, detection of even low copy numbers is achieved through automatic data analysis.
| Supplied in master mix |
QuantiFast Probe PCR Kit |
All cyclers from Applied Biosystems except Applied Biosystems 7500 |
| Supplied in a separate tube |
QuantiFast Probe PCR +ROX Vial Kit |
Applied Biosystems 7500, and cyclers from Agilent, Bio-Rad, Cepheid, QIAGEN, Eppendorf, Roche, and other suppliers |
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.
QuantiFast Probe Assays are predesigned, genomewide assays that use hydrolysis, probe-based detection. They are delivered with the QuantiFast Probe PCR Kit for guaranteed results in singleplex, two-step qRT-PCR.
Applications
QuantiFast Probe PCR Kits can be used for probe-based gene expression analysis of cDNA targets and 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.
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特点
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参数
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应用
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Probe-based, real-time PCR, two-step RT-PCR
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反应类型
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Real-time 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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cDNA, DNA
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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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Sequence-specific probes
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热循环仪
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All real-time cyclers (e.g. Roche LightCycler®, Corbett Rotor-Gene, Applied Biosystems)
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有/无ROX
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Available with ROX in master mix and with ROX as separate vial
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FAQ ID -1085
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For fast, quantitative, real-time PCR and two-step RT-PCR using sequence-specific probes
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图片
PCR反应时间极大缩短。
在标准PCR循环仪上使用QuantiFast Probe Kit进行两步法real-time RT-PCR,可缩短PCR总运行时间达60% (进行40个PCR循环;与标准的QIAGEN real-time PCR kit相比较)。L:LightCycler 2.0;A1:ABI PRISM 7900;A2:Applied Biosystems 7500;A3:ABI PRISM 7000。
线性范围广,灵敏度高。
在Mastercycler ep realplex上使用QuantiFast Probe PCR +ROX Vial Kit进行两步法PCR,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93%。使用10倍稀释的人白细胞cDNA和采用Primer Express设计的针对泛素 (一种调节蛋白) 的TaqMan Assay进行两次重复反应 (25 μl体积)。NTC:无模板对照。
更快获得结果,且保持高灵敏性。
在ABI PRISM 7900上使用10倍稀释的人白细胞cDNA (100 ng至0.01 ng) 和针对IL1RN (一种细胞因子) 的TaqMan Gene Expression Assay进行两次重复反应。QuantiFast Kit的灵敏度高于Supplier AII的标准循环试剂盒 (参照标准循环实验方案使用),具有更低的CT值,可实现低至10 pg cDNA的转录本定量。
灵敏的两步法RT-PCR。
在ABI PRISM 7900上使用10倍稀释的人白细胞cDNA (100 ng至0.01 ng) 和采用Primer Express设计的针对IL8 (一种趋化因子) 的TaqMan Assay进行三次重复反应。[A] QuantiFast Kit的灵敏度高于 [B] Supplier I的试剂盒 (参照标准循环实验方案使用),具有更低的CT值,可实现低至0.01 ng cDNA的转录本定量。
检测具有宽泛的动态范围。
在LightCycler 2.0上使用Supplier R提供的靶向b-2微球蛋白的一组引物和FRET探针,进行双重两步法RT-PCR反应。QuantiFast Kit的扩增曲线显示其具有较宽的动态范围。
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。
退火时引物快速结合。
[A] QuantiFast Buffer中的Q-Bond能使DNA聚合酶和引物结合为单一复合物,将退火时间缩短至几秒。此外,独特的缓冲液组分可支持DNA的熔解,缩短变性和延伸时间。[B] 若无Q-Bond,引物和聚合酶会相继与模板结合,退火时间较长。
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