基本描述

别名

超快速探针一步法 RT-qPCR U⁺ 试剂盒

英文名称

SuperFast Probe One Step RT-qPCR U⁺ Kit

储存温度

-20°C储存,避免反复冻融

运输条件

超低温冰袋运输

产品介绍

产品内容

S666097	Component	200 T	Storage
S666097A	5×SuperFast One Step RT-qPCR U+ Buffer	1 mL	-20℃. Avoid freeze/thaw cycle.
S666097B	SuperFast One Step U+ Enzyme	200 μL	-20℃. Avoid freeze/thaw cycle.
S666097C	RNase-Free Water	2×1.5 mL	-20℃. Avoid freeze/thaw cycle.

产品简介

SuperFast Probe One Step RT-qPCR U+ Kit专为以RNA为模板(如RNA病毒)的定量PCR检测而设计。使用基因特异性引物(GSP)，逆转录和qPCR反应在一管内完成，不需要额外的开管/移液操作，大大提高了检测通量，并降低了污染的风险。本试剂盒中引入了dUTP/UNG防污染系统。热敏感UNG在室温下即可将含U的污染物迅速降解；55℃逆转录时迅速失活，不会影响 qRT-PCR的效率和灵敏度。配合经过优化的缓冲体系和抗体修饰Taq酶以及突变后的M-MLV， SuperFast Probe One Step RT-qPCR U+ Kit的检测灵敏度可达到0.1pg总RNA或<10拷贝的RNA 模板且具有更高的热稳定性。5×SuperFast One Step RT-qPCR U+ Buffer包含优化的缓冲体系和 dNTP/dUTP Mix，尤其适用于TaqMan等荧光标记探针的高特异性、低模板浓度和多重快速检测。

注意事项

使用前请在本品完全融化后上下颠倒轻轻混匀，尽量避免起泡，并经短暂离心后使用。避免反复冻融本品。ROX染料用于校正定量PCR孔间产生的荧光信号误差，本品中不含ROX染料，如所使用仪器需匹配ROX染料。

PCR反应体系

试剂	50 μL体系	25 μL体系	终浓度
5×SuperFast One Step RT-qPCR U+ Buffer	10 μL	5 μL
SuperFast One Step U+ Enzyme	2 μL	1 μL	1×
Forward Primer，10 μM	1 μL	0.5 μL	0.2 μM1)
Reverse Primer，10 μM	1 μL	0.5 μL	0.2 μM
Probe2	0.5 μL	0.25 μL	0.1 μM
Template RNA3)	X μL	X μL
RNase-Free Water	Up to 50 μL	Up to 25 μL

注意：

1）通常引物终浓度以0.2μM可以得到较好结果，可以在0.1-1.0μM作为设定范围的参考。扩增效率不高的情况下，可提高引物的浓度；发生非特异性反应时，可降低引物浓度，由此优化反应体系。

2）所用探针的终浓度，与使用的荧光定量PCR仪、探针种类、荧光标记物质种类有关，实际使用时请参照仪器说明书，或各荧光探针的具体使用要求进行浓度的调节。

3）因不同物种的模板中含有的目的基因拷贝数不同，可对模板进行梯度稀释，以确定最佳的模板使用量

PCR反应条件

步骤	温度	时间	循环
逆转录	55℃	1 min	1
预变性	95℃	10s1)	1
变性	95℃	1 s	40-45
退火/延伸	55-60℃2)	10-15s3)	40-45

注意：

1）本产品所采用的酶在预变性95℃，30s条件下实现酶的活化。在此条件下，大多数模板可良好的进行解链。对GC含量高、二级结构复杂的模板，可将预变性时间延长至1min，以使起始模板充分解链，若高温处理时间过长，会对酶的活性造成影响；对于简单模板也可采用预变性1-10s，可根据模板情况确定最佳的预变性时间。

2）建议采用两步法PCR反应程序，退火温度请以55-60℃作为设定范围的参考，发生非特异性反应时，可提高退火温度。若因使用Tm值较低的引物或者扩增产物过长等原因，得不到良好的实验结果时，可尝试进行三步法PCR扩增。

3）实际使用的Real Time PCR仪是否支持快速扩增循环，初次尝试请进行预实验验证。

Products content

S666097	Component	200 T	Storage
S666097A	5×SuperFast One Step RT-qPCR U+ Buffer	1 mL	-20℃. Avoid freeze/thaw cycle.
S666097B	SuperFast One Step U+ Enzyme	200 μL	-20℃. Avoid freeze/thaw cycle.
S666097C	RNase-Free Water	2×1.5 mL	-20℃. Avoid freeze/thaw cycle.

Products Introduction

The SuperFast Probe One Step RT-qPCR U+ Kit is designed for quantitative PCR assays using RNA as a template (e.g., RNA viruses). Using gene-specific primers (GSP), reverse transcription and qPCR reactions are completed in a single tube, eliminating the need for additional tube-opening/pipetting operations, greatly increasing throughput and reducing the risk of contamination. The dUTP/UNG anti-contamination system is introduced in this kit. The heat-sensitive UNG rapidly degrades U-containing contaminants at room temperature; it is rapidly inactivated by reverse transcription at 55°C, without affecting the efficiency and sensitivity of qRT-PCR. Combined with optimized buffer systems and antibody-modified Taq enzymes and mutated M-MLV, the SuperFast Probe One Step RT-qPCR U+ Kit provides sensitivity up to 0.1 pg of total RNA or <10 copies of RNA template and enhanced thermal stability. 5× SuperFast One Step RT-qPCR U+ Buffer contains the following components The 5× SuperFast One Step RT-qPCR U+ Buffer contains an optimized buffer system and dNTP/dUTP Mix, which is particularly suitable for high specificity, low template concentration and multiplexed rapid detection of fluorescently labeled probes such as TaqMan.

caveat

Before use, please mix the product gently by turning it up and down after it is completely melted to avoid foaming, and use it after brief centrifugation. Avoid repeated freezing and thawing of the product.ROX dye is used to correct the fluorescence signal error between the quantitative PCR wells, this product does not contain ROX dye, if you need to match the ROX dye with the instrument you are using, please contact your local business or call CombiSense customer service at 4006-222-360.

PCR reaction system

Attention:

(1) Usually, the final primer concentration of 0.2 μM can get better results, and 0.1-1.0 μM can be used as a reference for setting the range. If the amplification efficiency is not high, the concentration of primer can be increased; if non-specific reaction occurs, the concentration of primer can be decreased to optimize the reaction system.

(2) The final concentration of the probe used is related to the fluorescence quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, please refer to the instrument manual or the specific requirements for the use of each fluorescent probe to adjust the concentration.

3) Because templates from different species contain different numbers of copies of the target gene, the template can be diluted in a gradient to determine the optimal amount of template to use

PCR reaction conditions

move	temp	timing	circulate
reverse transcription	55°C	1 min	1
premutability	95°C	10s1)	1
denaturation	95°C	1 s	40-45
Annealing/Extension	55-60°C2)	10-15s3)	40-45

Attention:

(1) The enzyme used in this product is activated under the condition of pre-denaturation at 95℃ for 30s. Under this condition, most of the templates can be well unchained. For templates with high GC content and complex secondary structure, the pre-denaturation time can be extended to 1min, so as to make the starting template fully unchained, and if the high temperature treatment time is too long, it will affect the activity of the enzyme; for simple templates, pre-denaturation time of 1-10s can also be used, and the optimal pre-denaturation time can be determined according to the template situation.

(2) It is recommended to use two-step PCR reaction program, the annealing temperature should be 55-60℃ as the reference range, and the annealing temperature can be increased when non-specific reaction occurs. If you can't get good results due to the use of primers with low Tm values or long amplification products, you can try three-step PCR amplification.

3) Whether the actual Real Time PCR instrument used supports rapid amplification cycles, please perform a pre-experiment to verify this for the first attempt.

质检证书（CoA,COO,BSE/TSE 和分析图谱)

C of A & Other Certificates(BSE/TSE, COO):

输入批号以搜索分析图谱:

技术文档和文章

superfast probe one step rt-qpcr u⁺ kit.pdf

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浓度 2 (C2)

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复溶计算器

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