QuantiFast SYBR® Green PCR Kit

使用SYBR Green I的快速两步法定量RT-PCR,适用于基因表达分析

  • 快速获得结果,可节省多达60%的时间
  • 可特异性检测低拷贝数的模板
  • 准确检测各种起始量的模板
  • 优化的即用型预混液用于快速PCR反应
  • 通用的操作流程适用于标准和快速PCR仪

QuantiFast SYBR Green PCR Kit应用SYBR Green I法两步法real-time RT-PCR,可对目标cDNA进行快速、特异性的定量检测。Q-Bond和优化的预混液可缩短real-time PCR反应时间,适用于标准或快速PCR仪。即用型预混液中的热启动酶和独特的PCR缓冲液可确保在所有real-time PCR仪上进行灵敏的qPCR,无需优化。为便于使用,预混液可保存在2–8°C。

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QuantiFast SYBR Green PCR Kit (4000)
For 4000 x 25 µl reactions: 2 x 25 ml 2x QuantiFast SYBR Green PCR Master Mix (contains ROX dye), 20 ml RNase-Free Water
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The QuantiFast 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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Significantly reduced PCR times.|Resolution of small differences in copy number.|Faster results without compromising sensitivity.|Sensitive two-step RT-PCR.|Specific primer annealing.|Fast primer annealing.|
The QuantiFast SYBR® Green PCR Kit reduces total PCR run time by up to 60% in real-time two-step RT-PCR on standard cyclers (40 cycles without melting curve analysis; comparison with standard QIAGEN real-time PCR kits). L: LightCycler 2.0; A1: ABI PRISM 7900; A2: Applied Biosystems 7500; A3: ABI PRISM 7000.|The QuantiFast SYBR® Green PCR Kit was used to detect the Y-chromosome-specific single-copy gene SRY in genomic DNA from a male donor using the Mastercycler ep realplex[A] Curves for 1000 down to 1.5 copies can be clearly distinguished from each other. [B] A plot of copy number (log) versus CT value demonstrates high linearity.|Expression of MYC (a proto-oncogene) in human leukocytes was analyzed by real-time two-step RT-PCR on the Applied Biosystems 7500 Fast System. Duplicate reactions were run using 10-fold cDNA dilutions (10 ng to 10 pg). [A] Fast cycling mode with the QuantiFast Kit gave similar CT values to [B] standard cycling mode with the QuantiTect Kit. In contrast, a kit for standard cycling from Supplier AII gave worse CT values not only in [C] fast cycling mode, but also in [D] recommended standard cycling mode.|Expression of MYC (a proto-oncogene) in human leukocytes was analyzed using the iCycler iQ and [A] the QuantiFast SYBR® Green PCR Kit or [B] a kit from Supplier Bv. Duplicate reactions were run using 10-fold cDNA dilutions (100 ng to 0.1 ng). The instrument-dedicated kit from Supplier BV was used according to the fast cycling protocol. The QuantiFast Kit gave lower CT values, indicating greater sensitivity.|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 SYBR Green PCR Kits通过快速PCR反应,实现了特异性、灵敏的real-time PCR检测(参见"Sensitive two-step RT-PCR")。PCR反应时间缩短60%(参见"Significantly reduced PCR times"),是您更快获得结果,而不影响PCR效果(参见"Faster results without compromising sensitivity")。您也可以大幅度提高样本通量,或与其他用户高效的共用PCR仪。

QuantiFast SYBR Green PCR Kit可清楚区分模板量的细微差别。即使低拷贝数的模板只有细微差别,该试剂盒也能准确定量(参见"Resolution of small differences in copy number")。

Principle

QuantiFast SYBR Green PCR Kits可在大范围内进行特异性、灵敏的检测,适用于标准和快速PCR仪。预混液中的SYBR Green I染料可分析多个目标核酸,无需合成序列特异性探针。特制的快速PCR缓冲液包含新型Q-Bond成分,可大大缩短变性、退火和延伸时间(参见"Fast primer annealing")。PCR缓冲液中平衡的K+和NH4+离子的配比可促进特异性的引物退火,确保高度灵敏和特异的PCR反应(参见"Specific primer annealing")。此外,HotStarTaq Plus DNA Polymerase必须要在95°C加热5分钟才能活化,需要严格的热启动,可避免生成非特异性产物。

2x QuantiFast SYBR Green PCR Kit的成分*
成分 特点 优势
HotStarTaq Plus DNA Polymerase 95ºC加热5分钟活化 在室温进行qPCR反应体系构建
QuantiFast SYBR Green PCR Buffer 平衡的NH4+和K+离子配比 引物探针的特异性结合确保获得可靠结果
独特的Q-Bond添加剂 PCR运行时间缩短,更快获得结果,一天内可完成更多PCR反应
SYBR Green I染料 与DNA双链结合时产生强荧光信号 高灵敏度扩增
ROX染料 对Applied Biosystems和Agilent PCR仪进行荧光信号的校准 对需要ROX染料的PCR仪进行校准,不影响PCR反应结果
* 也含有dNTP混合液(dATP、dCTP、dGTP和dTTP)。

Procedure

QuantiFast SYBR Green PCR Kit含有即用型预混液,无需对反应和扩增条件进行优化。只需在即用型PCR预混液中加入引物和cDNA模板,即可开始反应。按照操作手册中的操作流程,即可快速获得可靠的结果,适用于各种real-time PCR仪。

为获得最佳的两步法real-time RT-PCR结果,我们建议使用QuantiTect Reverse Transcription Kit合成cDNA。该试剂盒可在20分钟进行快速cDNA合成,并去除基因组DNA污染。

我们推荐适用QuantiTect Primer Assays进行SYBR Green法的基因表达分析。 QuantiTect Primer Assays是经生物信息学验证的引物,适用于人类、大鼠、小鼠和其他多个物种的全基因组。可直接在GeneGlobe订购该产品。

Applications

QuantiFast SYBR Green PCR Kits可用于cDNA的基因表达分析,适用于各种real-time PCR仪,包括Applied Biosystems、Bio-Rad、Cepheid、Eppendorf、Roche和Agilent的PCR仪。Rotor-Gene Q实时荧光定量PCR分析仪或其他Rotor-Gene PCR仪上使用时,我们推荐使用专为快速PCR研发的Rotor-Gene SYBR Green PCR Kit。

Feature
Specifications
Applications SYBR Green-based, real-time PCR, two-step RT-PCR
Reaction type Real-time and two-step RT-PCR
Real-time or endpoint Real-time
Sample/target type cDNA, DNA
Single or multiplex Single
SYBR Green I or sequence-specific probes SYBR Green I
Thermal cycler Applied Biosystems, Bio-Rad, Cepheid, QIAGEN, Eppendorf, Roche, and Agilent
With or without ROX With ROX
With/without hotstart With hotstart

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Kit Handbooks
2
For fast, quantitative, real-time PCR and two-step RT-PCR using SYBR Green I
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For genomewide, ready-to-use real-time RT-PCR assays using SYBR Green detection
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