酶系（Hotstart HiTaq& Script III）以其卓越的快速性及定量性被广泛应用，使用该产品进行Real Time RT-PCR反应可在同一反应管内连续进行，操作简单，并能有效防止污染。本制品中使用了 具有更高的热稳定性反转录酶（HS Script III RTase）和化学修饰热启动酶（Hotstart HiTaq DNA Polymerase），具有高扩增效率和高扩增特异性，能进行稳定的Real Time One Step RT-PCR反应，大大提高了检测灵敏度，并省略了PCR反应后的电泳步骤，非常适合于RNA病毒等微量RNA的检测。
本制品专为进行便利、高灵敏度的一步式RT-PCR设计。其独特的酶和特别设计的缓冲液确保了逆转录和PCR反应高效、准确的进行，无需进行优化。可以在宽广的定量区域内得到良好的标准曲 线，对靶基因进行准确定量检测，重复性好，可信度高。
适用范围：适用于RNA一步法扩增或热启动DNA扩增。
使用方法
1. 反应体系配制
1.1 溶解并混匀PCR反应所需的各种溶液，瞬时离心后，放置室温待用，探针建议避光处理。建议将PCR液体分装使用，避免反复冻融。
1.2 参照下表配制PCR反应体系：
组分
| 加入体积/25 μL体系
| 加入体积/50 μL体系 |
4× RT PCR Buffer
| 6.25 μL
| 12.5 μL |
酶系（Hotstart HiTaq& Script III）
| 1 μL
| 2 μL |
Primer-probe Mix
| 1 μL ※
| 2 μL ※ |
Templates
| 5 μL ※
| 10 μL ※ |
ddH₂O
| Up to 25 μL
| Up to 50 μL |
Total
| 25 μL
| 50 μL |
※注：Primer-probe Mix和Templates的用量可自行调整。
1.3 用移液器轻轻吹打混匀或轻微Vortex混匀，室温离心数秒，使液体聚于管底。
1.4 将各PCR反应管置于设置好的PCR仪上，开始PCR反应。
2. 反应程序
注：
(1) 通常各引物使用的终浓度为0.2 μM，引物的Tm值一般设定在55℃以上，可借助相关软件进行分析，使用理想的引物浓度和退火温度可以得到理想的反应效果。
(2) 对于大多数实验，45个循环数是足够的，若您的样本本身靶标含量较高，可适当减少循环数如40个循环，缩短检测时间。
Enzyme System (Hotstart HiTaq & Script III), renowned for its exceptional speed and quantitative performance, is widely used. Real Time RT-PCR reactions using this product can be performed consecutively within the same reaction tube, offering simple operation and effective contamination prevention. This product incorporates a reverse transcriptase with higher thermal stability (HS Script III RTase) and a chemically modified hot-start enzyme (Hotstart HiTaq DNA Polymerase), providing high amplification efficiency and specificity. It enables stable Real Time One-Step RT-PCR reactions, significantly improves detection sensitivity, and eliminates the need for post-PCR electrophoresis. It is particularly suitable for detecting low-abundance RNA, such as from RNA viruses.
This product is specifically designed for convenient and highly sensitive One-Step RT-PCR. Its unique enzyme system and specially formulated buffer ensure efficient and accurate reverse transcription and PCR reactions without the need for optimization. It yields excellent standard curves over a wide quantitative range, allowing for accurate quantitative detection of target genes with good repeatability and high reliability.
Scope of Application: Suitable for One-Step amplification of RNA or hot-start DNA amplification.
Instructions for Use
1. Preparation of Reaction Mixture
1.1 Thaw and mix all solutions required for the PCR reaction. After a brief centrifugation, place them at room temperature for use. Probes should be protected from light. It is recommended to aliquot PCR reagents to avoid repeated freeze-thaw cycles.
1.2 Prepare the PCR reaction mixture according to the table below:
Component
| Volume for 25 µL System
| Volume for 50 µL System
|
4× RT PCR Buffer
| 6.25 μL
| 12.5 μL |
Enzyme System (Hotstart HiTaq & Script III)
| 1 μL
| 2 μL |
Primer-probe Mix
| 1 μL ※
| 2 μL ※ |
Templates
| 5 μL ※
| 10 μL ※ |
ddH₂O
| Up to 25 μL
| Up to 50 μL |
Total
| 25 μL
| 50 μL |
※ Note: The amounts of Primer-probe Mix and Templates can be adjusted as needed.
1.3 Mix gently by pipetting or by brief vortexing. Centrifuge briefly at room temperature to collect all liquid at the bottom of the tube.
1.4 Place the PCR reaction tubes on a pre-programmed thermal cycler and start the PCR reaction.
2. Reaction Protocol
Notes:
(1) The typical final concentration for each primer is 0.2 µM. The Tm value of the primers is generally recommended to be set above 55°C. Analysis with relevant software is advised. Using optimal primer concentrations and annealing temperatures will yield ideal reaction results.
(2) For most experiments, 45 cycles are sufficient. If your samples have a high target content, the cycle number can be appropriately reduced (e.g., to 40 cycles) to shorten the detection time.
ZJ26F0434224
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