Menu ccc

RapiDxFire™ Thermostable Reverse Transcriptase

Cat.No. Size Price Quantity Subtotal Buy
30250-1 50 rxns 來電價
30250-2 250 rxns 來電價

Products Description


一種真正的熱穩定反轉錄酶,用於快速合成短cDNA(<1 Kb)

  • 在高溫(55至80°C)下非常活躍:可提高cDNA合成的特異性
  • 敏感: 在兩步驟的RT-qPCR分析中可檢測≤100 copies的RNA
  • 反應時間短(5分鐘或更短): 可簡化RT-qPCR工作流程和更快得到結果
  • 在室溫下活性穩定(> 3個月):簡化自動化的設置(可無需冷藏),可在無法冷藏的環境中使用
  • Lyo兼容:不含甘油和其他成分 已知會干擾下游凍乾的成分
  • 批次間重複性:在ISO 13485認證的生產設施製造

RapiDxFire™熱穩定逆轉錄酶是一種顯著優於常見的MMLV和AMV逆轉錄酶。從溫泉中鑑定分離出的噬菌體酵素,在升高的溫度下表現出越來越高的活性(不但在90℃下10分鐘後尚可保持約60%活性)並且在20-25℃下儲存> 3個月後保持其活性。這種酶缺乏RNAse H和3’ to 5’ exonuclease activity 活性並有效合成短cDNA片段(≤1Kb)。與其他反轉錄酶一樣,RapiDxFire RT具有DNA聚合酶活性,但缺乏5'3'核酸外切酶活性。在不含甘油,不含Triton™X-100的儲存緩衝液,可進一步優化用於下游凍乾。


Figure 1. Reaction temperature profile for RapiDxFire Thermostable Reverse Transcriptase. Reverse transcription reactions were set up on ice using each manufacturer’s recommended buffer system and Poly (rC) /p(dG)12-18 template/primer substrate. Reactions were transferred from ice to the indicated temperatures (37, 50, 55, 60, 65, 70, 75, and 80°C) and incubated for 40 minutes. Following incubation, RNA/cDNA heteroduplex product is quantified as a measure of polymerization activity, utilizing PicoGreen® fluorescence on a Tecan Infinite® M1000 Pro. RapiDxFire Thermostable RT exhibits increasing activity as the reaction temperature is increased, up to 80ºC (highest temperature tested); ~60% activity remains after 10 min at 90⁰C (data not shown).

用RapiDxFire Thermostable RT檢測茲卡病毒優異表現

Figure 2. cDNA synthesis time course studies in a 2-step RT-qPCR reaction with different thermostable RTs. A) qPCR curve after a 1 minute reverse transcription reaction. Zika cDNA synthesis was conducted for each enzyme in duplicate using target-specific primers and recommended reaction buffer and incubation temperatures per suppliers’ guidelines (RapiDxFire= 60⁰C, Supplier B= 50⁰C, and Supplier T= 55⁰C). After cDNA synthesis real time PCR was performed using one-tenth volume of each of the cDNA samples. PCR was performed using EconoTaq and its supplied buffer (Lucigen Cat No. 30031-3). Detection of PCR 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) Reverse transcription time course study prior to performing second-step real-time PCR. Encircled data points are derived from data represented in the qPCR curve in A).


Figure 3. RapiDxFire Thermostable RT was stored in separate aliquots per time point at ambient and -20°C. At each time point RapiDxFire Thermostable RT was evaluated by measuring first-strand cDNA synthesis of 10000 copies MS2 RNA. cDNA synthesis was carried out using a gene-specific primer for MS2 at reaction temperature of 60°C for 5 minutes. cDNA was measured using real time PCR and MS2 primers design around a 520 base pair amplicon.



Contents include RapiDxFire Thermostable Reverse Transcriptase (3 Units/ µL) and 10X RapiDxFire Reaction Buffer. The enzyme pack sizes (50 or 250 Rxns) are based on 25 µL reaction volumes.