基本描述

别名

HyperProbe 混合物

英文名称

HyperProbe Mixture

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

H666048	Component	5 mL	Storage
H666048A	2×HyperProbe Mixture	5×1 mL	-20℃. Avoid freeze/thaw cycle.
H666048B	ddH2O	5×1 mL	-20℃. Avoid freeze/thaw cycle.

产品简介

HyperProbe Mixture是专用于探针法(TaqMan，Molecular Beacon等）进行实时荧光定量PCR的预混体系，浓度为2×，包含新型工程DNA酶、PCR Buffer、dNTPs、 Mg2+以及增强剂和稳定剂，操作简单方便。主要用于基因组DNA靶序列和RNA反转录后的cDNA靶序列检测。本品含有高灵敏度工程DNA酶，能在有效减少常温条件下由引物和模板非特异性结合或引物二聚体而产生的非特异性扩增的同时，大幅提升检测灵敏度和扩增效率，酶的激活仅需在95℃孵育30 s，大大缩短了PCR的反应时间。优化的PCR缓冲体系与混合酶的组合，有效抑制了非特异产物的产生，能够显著提高PCR的扩增效率，荧光信号更强，灵敏度更高。

注意事项

1.使用前请上下颠倒轻轻混匀，尽量避免起泡，并经短暂离心后使用。

2.避免反复冻融本品，反复冻融可能使产品性能下降。本产品长期保存可置于-20℃ 避光保存。如果在短期内需要频繁使用，可在2-8℃保存。

3.ROX染料用于校正定量PCR仪孔与孔之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。

注意事项

1．使用前请上下颠倒轻轻混匀，尽量避免起泡，并经短暂离心后使用。

2．本产品中含有荧光染料SYBR Green I，保存本产品或配制PCR反应液时应避免强光照射。

3．避免反复冻融本品，反复冻融可能使产品性能下降。本产品长期保存可置于-20℃，避光。如果在短期内需要频繁使用，可在2-8℃保存。

4．本品不能用于探针法荧光定量PCR。

使用方法

以下举例为常规PCR反应体系和反应条件，实际操作中应根据模板、引物结构和目的片段大小不同进行相应的改进和优化。

PCR反应体系

试剂	50 μl体系	25 μl体系	20 μl体系	终浓度
2×HyperProbe Mixture	25 μl	12.5 μl	10 μl	1×
Forward Primer，10 μM Reverse Primer，10 μM Probe2)	1 μl 1 μl 1 μl	0.5 μl 0.5 μl 0.5 μl	0.4 μl 0.4 μl 0.4 μl	0.2 μM1) 0.2 μM1) 0.2 μM
Template DNA3)	X μl	X μl	X μl
ddH2O	up to 50 μl	up to 25 μl	up to 20 μl

注意：

1）通常引物浓度以0.2 M可以得到较好结果，可以在0.1-1.0 μM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）通常DNA模板的量以10-100 ng基因组DNA或1-10 ng cDNA为参照，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。

2. PCR反应条件

步骤	温度	时间
预变性	95℃	30s1）
变性	95℃	10 s	40-45 个循环数
退火/延伸	58℃	20 s2）	40-45 个循环数

注意：

1）本产品所采用的酶在预变性95℃、30 s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1分钟，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20 s，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58-64℃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，退火温度请以56℃-64℃的范围作为设定参考。几种常见仪器的退火延伸时间设定见下表：使用Roche，BioRad、Agilent和宏时、东胜龙等公司荧光定量PCR仪时请设定在20 s。使用ABI 7000/7300/7500时请设定在30 s。退火/延伸时间可根据使用不同型号仪器和不同模板进行设定，请按照仪器使用说明书要求进行实验操作。

Products Content:

H666048	Component	5 mL	Storage
H666048A	2×HyperProbe Mixture	5×1 mL	-20℃. Avoid freeze/thaw cycle.
H666048B	ddH2O	5×1 mL	-20℃. Avoid freeze/thaw cycle.

Products Introduction

HyperProbe Mixture is a premixed system for real-time fluorescence quantitative PCR by probe method (TaqMan, Molecular Beacon, etc.). The concentration is 2×, which contains a new engineered DNA enzyme, PCR Buffer, dNTPs, Mg2+, enhancers and stabilizers, and it is easy to operate. It is mainly used for the detection of genomic DNA target sequences and cDNA target sequences after RNA reverse transcription.

This product contains highly sensitive engineered DNA enzyme, which can effectively reduce the non-specific amplification caused by the non-specific binding of primers and templates or primer dimer at room temperature, and at the same time, greatly improve the detection sensitivity and amplification efficiency, and the activation of the enzyme only needs to be incubated at 95 ℃ for 30 s, which greatly shortens the reaction time of PCR. The optimized PCR buffer system and enzyme mixture effectively inhibit the generation of non-specific products, which can significantly improve the PCR amplification efficiency, stronger fluorescence signal and higher sensitivity.

caveat

1. Before use, mix gently by turning up and down, avoid foaming as much as possible, and use after brief centrifugation.

2. Avoid repeated freezing and thawing of the product, which may degrade the performance of the product. This product can be stored at -20℃ for long term storage and protected from light. If frequent use is required within a short period of time, it can be stored at 2-8℃.

3. ROX dye is used to correct the fluorescence signal error generated between the wells of the quantitative PCR instrument, ROX is not contained in this product.

If you need to match ROX dyes with your instrument, please contact your local sales office or call CombiSense customer service.

4006-222-360.

Usage

The following examples are conventional PCR reaction systems and conditions, which should be improved and optimized according to the template, primer structure and target fragment size.

PCR reaction system

Attention:

(1) Usually, a primer concentration of 0.2 M can give 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 manual of the instrument or the specific requirements for the use of each fluorescent probe to adjust the concentration.

3) Usually the amount of DNA template is 10-100 ng of genomic DNA or 1-10 ng of cDNA as a reference, as templates of different species

The number of copies of the target gene contained in them varies, and a gradient dilution of the template can be performed to determine the optimal amount of template to use.

PCR reaction conditions

Attention:

(1) The enzyme used in this product is activated by pre-denaturation at 95°C for 30 s. Most of the templates can be deconvoluted well under this condition. 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 1 minute, so that the starting template can be 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 can also be used for 20 s, and the optimal pre-denaturation time can be determined according to the template situation.

(2) It is recommended to use two-step PCR program, the annealing temperature should be 58-64℃ 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 value, you can try three-step PCR amplification, and the annealing temperature should be set in the range of 56℃-64℃ as a reference. The annealing and extension times for several common instruments are shown in the following table: 20 s for Roche, BioRad, Agilent, Hongshi, Dongshenglong, etc. 30 s for ABI 7000/7300/7500. The annealing/extension times can be set according to the different types of instruments and templates, please follow the instructions of the instruments. The annealing/extension time can be set according to different models of instruments and templates, please follow the instruction manual of the instrument.

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

hyperprobe mixture.pdf

溶液计算器

摩尔浓度计算器

计算溶液所需的质量、体积或浓度。

质量

=

浓度

x

体积

x

分子量

g/mol

稀释计算器

计算配制储备溶液所需的稀释度。

浓度1（C1）

x

体积 1 (V1)

=

浓度 2 (C2)

x

体积 2 (V2)

复溶计算器

复溶计算器允许您快速计算试剂的体积，以复溶小瓶。只需输入试剂的质量和目标浓度，计算器将确定其余部分。

体积（添加到小瓶中）

=

质量（小瓶）

÷

所需的复溶浓度