Hotstart HiTaq& Super M-MuLV One Step RT-qPCR Kit (with UDG) 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。酶系使用热敏型的UDG酶，搭配含U的底物，可以有效防止上一次的PCR产物对本次实验额污染，同时热敏型的UDG酶在50℃的条件下，完全失去活性，不影响本次实验的逆转录产物。
本试剂中使用了稳定、高效的反转录酶（Super M-MuLV Reverse Transcriptase）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，非常适合于微量RNA的检测。
本试剂专为进行便利、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：以 RNA 为模板的核酸定量或定性检测，逆转录+扩增在一管内完成，无需开盖。
组分表
FP1509101 | 组分 | 100T | 1000T | 10000T | 存储温度 |
FP1509101A | Hotstart HiTaq& Super M-MuLV Mix | 200 μL | 2×1.0 mL | 20 mL | -20℃ |
FP1509101B | Buffer Mix | 950 μL | 10×0.95 mL | 95 mL | -20℃ |
使用说明：
1、反应体系配制：
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 PCR 液体分装使用，避免反复冻融。
2、参照下表配制 PCR 反应体系：
组分
| 加入体积/25μL 体系
| 加入体积/50μL 体系
| 终浓度
|
Buffer mix
| 9.5 μL
| 19 μL
| 1×
|
酶系（Hotstart HiTaq& Super M-MuLV）
| 2μL
| 4 μL
| 2/50 μL
|
引物探针
| 1 μL
| 2 μL
| / |
模板
| 5 μL
| 10 μL
| /
|
ddH₂O | To 25 μL
| To 50 μL
| /
|
终体积
| 25 μL
| 50 μL
|
|
注：引物、探针以及模板的用量可自行调整。
3、用移液器轻轻吹打混匀或轻微 Vortex 混匀，室温离心数秒，使液体积聚于管底。
4、将各 PCR 反应管置于 PCR 仪上，开始 PCR 反应。
步骤名称
| 温度
| 时间 | 循环 |
逆转录
| 50℃
| 15min
| 1 |
预变性
| 95℃
| 10min
| 1 |
变性
| 95℃
| 10s
| 45 |
退火
| 60℃
| 40s
| 45
|
保存
| 12℃
| ∞
| 1 |
注：反应条件可根据实际情况自行调整。
Hotstart HiTaq& Super M-MuLV One Step RT-qPCR Kit (with UDG) is widely recognized for its exceptional speed and quantitative accuracy. This product enables continuous Real-Time RT-PCR reactions within a single tube, simplifying operations. The enzyme system employs thermolabile UDG (Uracil-DNA Glycosylase) in combination with dUTP-containing substrates, effectively preventing carryover contamination from previous PCR products. The thermolabile UDG is completely inactivated at 50°C, ensuring it does not interfere with the reverse transcription products of the current reaction.
This kit utilizes a highly stable and efficient reverse transcriptase (Super M-MuLV Reverse Transcriptase) and a chemically modified hot-start DNA polymerase (Hotstart HiTaq DNA Polymerase). It delivers high amplification efficiency and specificity, enabling robust Real-Time One-Step RT-PCR. This significantly enhances detection sensitivity and eliminates the need for post-PCR electrophoresis, making it particularly suitable for the detection of trace amounts of RNA.
Specifically designed for convenient and highly sensitive one-step RT-PCR, this kit features a unique enzyme formulation and specially optimized buffer system. These ensure efficient and accurate reverse transcription and PCR amplification without the need for reaction optimization. It generates excellent standard curves over a wide quantitative range, allowing precise quantitative detection of target genes with high reproducibility and reliability.
Applications: Quantitative or qualitative detection of nucleic acids using RNA as template. Reverse transcription and amplification are completed in a single tube without tube opening.
Product Component List
FP1509101 | Components | 100T | 1000T | 10000T | Storage |
FP1509101A | Hotstart HiTaq& Super M-MuLV Mix | 200 μL | 2×1.0 mL | 20 mL | -20℃ |
FP1509101B | Buffer Mix | 950 μL | 10×0.95 mL | 95 mL | -20℃ |
Protocol:
1. Reaction Setup:
Thaw and thoroughly mix all PCR solutions required for the reaction.
Briefly centrifuge the solutions and keep them at room temperature until use.
Probes should be protected from light.
Recommendation: Aliquot PCR reagents to avoid repeated freeze-thaw cycles.
2. Prepare the PCR Reaction Mixture according to the table below:
Component
| Volume/25 μL
| Volume/50 μL
| Final Concentration
|
Buffer Mix
| 9.5 μL
| 19 μL
| 1×
|
Enzyme Mix (Hotstart HiTaq& Super M-MuLV)
| 2 μL
| 4 μL
| 2/50 μL
|
Primer-Probe Mix
| 1 μL
| 2 μL
| / |
Template
| 5 μL
| 10 μL
| /
|
ddH₂O | To 25 μL
| To 50 μL
| /
|
Total Volume
| 25 μL
| 50 μL
|
|
Note: The amounts of primers, probes, and template can be adjusted according to experimental requirements.
3. Mixing:
Gently mix the reaction components by pipetting up and down or by brief vortexing. Centrifuge briefly at room temperature to collect the liquid at the bottom of the tube.
4. PCR Amplification:
Place the PCR reaction tubes in the thermal cycler and initiate the PCR program.
Standard Thermal Cycling Protocol:
Step
| Temperature
| Time
| Cycles
|
Reverse Transcription
| 50℃
| 15min
| 1 |
Initial Denaturation
| 95℃
| 10min
| 1 |
Denaturation
| 95℃
| 10s
| 45 |
Annealing/Extension
| 60℃
| 40s
| 45
|
Hold
| 12℃
| ∞
| 1 |
Note: Reaction conditions can be adjusted based on specific experimental requirements.
ZJ26F0434240
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