Fast Probe Mixture

产品内容

产品简介

Fast Probe Mixture是专用于探针法(TaqMan，Molecular  Beacon等）实时荧光定量PCR的预混体系，浓度为2×,包含Fast Taq DNA Polymerase、PCR Buffer、dNTPs、Mg2+等，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。本品含有的Fast Taq DNA Polymerase，能有效减少在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，酶的激活仅需在95℃  孵育30s。整个PCR反应过程比普通反应可节省约40分钟，大大缩短了PCR的反应时间。独特的PCR缓冲体系与快速热启动酶的组合，有效抑制了非特异产物的产生，并显著提高了PCR的扩增效率，荧光信号更强，灵敏度更高，线性范围更宽。该产品适用范围广，可用于普通和快速定量PCR程序。

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，一般用于ABI、Stratagene等公司的Real Time PCR 扩增仪。不同仪器的激发光学系统有所不同，因此ROX染料的浓度必须与相应的荧光定量PCR仪相匹配。

不需要ROX校正的仪器（F665766）：

Roche LightCycler 480，Roche LightCyler 96，Bio-rad iCyler iQ，iQ5，CFX96等。

Low ROX校正的仪器（F665768）：

ABI Prism7500/7500 Fast，QuantStudio® 3 System，QuantStudio® 5 System， QuantStudio® 6 Flex System，QuantStudio® 7 Flex System，ViiA 7 system， Stratagene Mx3000/Mx3005P，Corbett Rotor Gene 3000等。

需要High ROX校正的仪器（F665774）：

ABI Prism7000/7300/7700/7900，Eppendorf，ABI Step One/Step One Plus等。

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。如果在短期内需要频繁使用，可在2-8℃保存。

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。 

1. PCR反应体系

注意：1）通常引物浓度以0.2 μM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。

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。

4） 不同仪器的激发光学系统有所不同，根据使用荧光定量的仪器选择加入50× Low ROX or 50×High ROX。

2. PCR反应程序： 

建议采用两步法PCR反应程序，本程序是以ABI 7500荧光定量PCR仪为参照设定。

注意：1）本产品所采用的酶须在预变性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1-4分钟，以使起始模板充分解链。 

2）建议采用两步法PCR反应程序，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。

Product content

Product Introduction

Fast Probe Mixture is a pre-mixed system for real-time fluorescence PCR by probe method (TaqMan, Molecular Beacon, etc.), with a concentration of 2×, including Fast Taq DNA Polymerase, PCR Buffer, dNTPs, Mg2+ and so on, which is easy and convenient to operate. It is mainly used for the detection of genomic DNA target sequence and cDNA target sequence after RNA reverse transcription. The Fast Taq DNA Polymerase contained in this product can effectively reduce the non-specific amplification generated by the non-specific binding of primers and templates or primer dimerization at room temperature, and the activation of the enzyme only needs to be incubated at 95 ℃ for 30 s. The whole PCR reaction process can save about 40 minutes compared with the ordinary reaction, which greatly shortens the reaction time of PCR. The combination of unique PCR buffer system and fast hot start enzyme effectively inhibits the generation of non-specific products and significantly improves the PCR amplification efficiency with stronger fluorescence signal, higher sensitivity and wider linear range. The product has a wide range of applications and can be used for both normal and rapid quantitative PCR programs.

ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used in Real Time PCR amplifiers from ABI, Stratagene, and other companies. The excitation optics vary from instrument to instrument, so the concentration of ROX dye must be matched to the corresponding fluorescence quantitative PCR instrument.

Instruments that do not require ROX calibration (F665766):

Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96 and others.

Instruments that require Low ROX calibration (F665768):

ABI Prism7500/7500 Fast, QuantStudio®3 System, QuantStudio®5 System, QuantStudio®6 Flex System, QuantStudio®7 Flex System, ViiA 7 system. Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, and more.

Instruments that require High ROX calibration (F665774):

ABI Prism 7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, and others.

matters needing attention

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

Usage

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

1.PCR reaction system

Note: 1) Usually the primer concentration of 0.2μM can get better results, and 0.1-1.0μM can be used as a reference for setting the range. 2) The final concentration of the probe used is related to the fluorescent quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, so please refer to the instruction manual of the instrument or the specific requirements of the use of each fluorescent probe for the adjustment of the concentration in actual use.

(3) Usually the amount of DNA template is 10-100ng genomic DNA or 1-10ng cDNA as a reference. Since the templates of different species contain different copy numbers of target genes, the templates can be subjected to gradient dilution to determine the optimal amount of template to be used.

(4) The excitation optical system varies from instrument to instrument, choose to add 50×Low ROX or 50×High ROX according to the instrument using fluorescence quantification.

2. PCR reaction program:

A two-step PCR reaction program is recommended, and this program is set up using the ABI 7500 Fluorescent Quantitative PCR Instrument as a reference.

Note: 1) The enzyme used in this product must be pre-denatured at 95°C for 30s to achieve enzyme activation. 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 1-4 minutes in order to make the starting template fully unchained.

(2) It is recommended to use two-step PCR reaction program, if you can not get good experimental results due to the use of primers with lower Tm values, etc., you can try to carry out a three-step PCR amplification, annealing temperature, please use the range of 56 ℃ - 64 ℃ for as a reference for the setting.