RapiDxFire™ Thermostable Reverse Transcriptase 热稳定逆转录酶

一种真正的热稳定逆转录酶，用于快速合成短cDNA（<1 Kb） (30250-1  30250-2)

• 在高温下极其活跃（55-80°C）：通过各种RNA模板提高cDNA合成的特异性
• 敏感： 在两步RT-qPCR分析中检测≤100拷贝的RNA
• 反应时间短（5分钟或更短）： 简化的RT-qPCR工作流程和更快的出结果时间
• 在室温下稳定（> 3个月）：简化自动化卡座上的设置（无需冷藏），可在冷藏有限的环境中使用
• Lyo兼容： 酶制剂不含甘油和其他成分，不会干扰下游冻干步骤
• 批次间重复性：在ISO 13485认证的设施中制造。

产品包含RapiDxFireThermostable Reverse Transcriptase（3 Units /μL）和10XRapiDxFire Reaction Buffer。
酶包装大小（50或250 Rxns）基于25μL反应体积。
         RapiDxFire热稳定逆转录酶是一种显着优于流行的MMLV和AMV逆转录酶的方法。最初在温泉中发现，这种强大的噬菌体酶在升高的温度下表现出越来越高的活性（在90℃下10分钟后活性保持约60％）并且在20-25℃下储存> 3个月后保持其核心活性。这种酶缺乏RNAse H和3'→5'核酸外切酶活性并有效合成短cDNA片段（≤1Kb）。与其他逆转录酶一样，RapiDxFire RT具有DNA聚合酶活性，但缺乏5'→3'核酸外切酶活性。酶的储存缓冲液不含甘油和Triton™X-100，可进一步优化用于下游冻干步骤。
RapiDxFire耐高温RT在高温下表现最佳
  Figure 1. Reaction temperature profile for RapiDxFireThermostable Reverse Transcriptase. Reverse transcriptionreactions were set up on ice using each manufacturer’s recommended buffersystem and Poly (rC) /p(dG)12-18 template/primer substrate. Reactions weretransferred from ice to the indicated temperatures (37, 50, 55, 60, 65, 70, 75,and 80°C) and incubated for 40 minutes. Following incubation, RNA/cDNAheteroduplex product is quantified as a measure of polymerization activity,utilizing PicoGreen® fluorescence on a Tecan Infinite® M1000 Pro. RapiDxFire ThermostableRT exhibits increasing activity as the reaction temperature is increased, up to80ºC (highest temperature tested); ~60% activity remains after 10 min at 90⁰C(data not shown).
  Figure 2. cDNA synthesistime course studies in a 2-step RT-qPCR reaction with different thermostableRTs. A) qPCR curve after a 1 minute reversetranscription reaction. Zika cDNA synthesis was conducted for each enzyme induplicate using target-specific primers and recommended reaction buffer andincubation temperatures per suppliers’ guidelines (RapiDxFire= 60⁰C, SupplierB= 50⁰C, and Supplier T= 55⁰C). After cDNA synthesis real time PCR wasperformed using one-tenth volume of each of the cDNA samples. PCR was performedusing EconoTaq and its supplied buffer (Lucigen Cat No. 30031-3). Detection ofPCR products were done real time using an intercalating dye (Dyomics Cat No. V13-01184)and BioRad CFX C1000 Touch™ with absorption/emission of 481nm/526nm. B) Reversetranscription time course study prior to performing second-step real-time PCR.Encircled data points are derived from data represented in the qPCR curve inA).

3个月室温稳定性  图3. RapiDxFire热稳定性室温环境和-20°C下每个时间点单独存储活性对比。 在每个时间点RapiDxFire Thermostable RT通过测量10000拷贝MS2 RNA的第一链cDNA合成来评估。使用MS2的基因特异性引物在60℃的反应温度下进行cDNA合成5分钟。使用实时PCR和围绕520碱基对扩增子设计的MS2引物测量cDNA。

产品名称： RapiDxFire Thermostable ReverseTranscriptase, 3Units/µL
规格：50次， 250次 
对应货号： 30250-1  30250-2
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