Products Description
RapiDxFire™熱穩定反轉錄酶
一種真正的熱穩定反轉錄酶,用於快速合成短cDNA(<1 Kb)
特色:
- 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。
RapiDxFire耐高溫RT在高溫下表現最佳
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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
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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). |
3個月室溫穩定性
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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.
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Information
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.
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