计算溶液所需的质量、体积或浓度。
为什么选择此级别
，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
-20°C储存,避免反复冻融。超低温运输 。请查阅批次 COA 获取详细规格。
质量文档
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文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
产品内容
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产品简介
2×GoldStar Probe Mixture（UNG）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GoldStar Taq DNA polymerase、 PCR Buffer、dNTPs（dTTP全部被dUTP所取代）、 UNG酶和Mg2+ ，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靶序列的检测，如基因表达分析， 拷贝数分析， SNP基因型分析等。本产品中运用了dUTP-UNG防污染系统，在PCR反 应体系配制过程中加入了dUTP，因此就形成了含有dU碱基的扩增产物。而此产物可以 在下次进行PCR反应前，由PCR体系中的UNG酶处理消除。这样就有效的去除了PCR 产物的残留污染，大大降低了由于扩增产物污染而导致的假阳性。 UNG酶在PCR循环 中的预变性步骤即可被灭活，因此不会影响新的含dU碱基PCR产物的形成。本品含有 的GoldStar Taq DNA Polymerase是一种经化学修饰的、全新高效热启动酶，在常温下 没有聚合酶活性，有效避免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，酶的激活须在95℃下孵育10分钟。独特的PCR缓冲体系与热启动 酶的组合，显著提高了PCR的扩增效率，荧光信号更强，灵敏度更高，可以检测单拷贝 的模板。使用本产品可以得到更广的线性范围，对目的基因定量更准确。
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（G670150 ） ：
Roche LightCycler 480，Roche LightCyler 96，Bio-rad iCyler iQ，iQ5，CFX96等。
需要Low ROX校正的仪器（G665780） ：
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（G665787） ：
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
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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 ）使用的探针浓度，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
注意！本产品预变性反应必须在95℃ 10分钟下完成！
两步法PCR：
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注意：
1 ）本产品所采用的热启动酶须在预变性95 ℃、 10 min条件下实现酶的活化。
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。
三步法PCR：
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Product content
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The 2x GoldStar Probe Mixture (UNG) is a pre mixed system specifically designed for real-time fluorescence quantitative PCR using probe methods such as TaqMan and Molecular Beacon. It has a concentration of 2x and includes GoldStar Taq DNA polymerase, PCR buffer, dNTPs (all dTTPs are replaced by dUTP), UNG enzyme, and Mg2+. The operation is simple and convenient. Mainly used for detecting genomic DNA target sequences and cDNA target sequences after RNA reverse transcription, such as gene expression analysis, copy number analysis, SNP genotype analysis, etc. This product utilizes the dUTP-UNG pollution prevention system, and dUTP is added to the PCR reaction system during the preparation process, resulting in the formation of amplification products containing dU bases. And this product can be eliminated by UNG enzyme treatment in the PCR system before the next PCR reaction. This effectively removes residual contamination of PCR products and greatly reduces false positives caused by amplification product contamination. The pre denaturation step of UNG enzyme in the PCR cycle can be inactivated, so it will not affect the formation of new PCR products containing dU bases. The GoldStar Taq DNA Polymerase contained in this product is a chemically modified, novel, and highly efficient hot start enzyme. It has no polymerase activity at room temperature, effectively avoiding non-specific amplification caused by non-specific binding of primers and templates or primer dimerization at room temperature. Enzyme activation requires incubation at 95 ℃ for 10 minutes. The unique combination of PCR buffer system and hot start enzyme significantly improves the amplification efficiency of PCR, with stronger fluorescence signal and higher sensitivity, which can detect single copy templates. By using this product, a wider linear range can be obtained, resulting in more accurate quantification of the target gene.
ROX dye is used to correct the fluorescence signal error between wells in quantitative PCR instruments, and is generally used in Real Time PCR amplification instruments from companies such as ABI and Stratagene. The excitation optical systems of different instruments vary, so the concentration of ROX dye must be matched with the corresponding fluorescence quantitative PCR instrument.
Instruments that do not require ROX calibration (G670150) :
Roche LightCyster 480, Roche LightCyler 96, Bio rad iCyler iQ, iQ5, CFX96, etc.
Instruments that require Low ROX calibration (G665780) :
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, etc.
Instruments that require High ROX calibration (G665787) :
ABI Prism7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, etc.
matters needing attention
1. Before use, please gently mix upside down to avoid foaming, and use after briefly centrifugation.
2. Avoid repeated freezing and thawing of this product, as repeated freezing and thawing may cause a decrease in product performance. This product can be stored for a long time at -20 ℃, away from light. If frequent use is required in the short term, it can be stored at 2-8 ℃.
Usage
The following are examples of conventional PCR reaction systems and reaction conditions. In practical operation, corresponding improvements and optimizations should be made based on different templates, primer structures, and target fragment sizes.
1. PCR reaction system
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Attention:
1) Typically, the primer concentration is 0.2 μ M can achieve good results, ranging from 0.1 to 1.0 μ M serves as a reference for setting the range.
2) The 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. Please refer to the instrument manual or the specific usage requirements of each fluorescent probe for concentration adjustment during actual use.
3) The amount of DNA template is usually based on 10-100 ng genomic DNA or 1-10 ng cDNA as a reference. Due to the different copy numbers of target genes contained in templates of different species, gradient dilution can be applied to the template to determine the optimal template usage.
4) The excitation optical systems of different instruments vary, and depending on the instrument used for fluorescence quantification, 50 x Low ROX or 50 x High ROX can be added.
2. PCR reaction program
Attention! The pre denaturation reaction of this product must be completed at 95 ℃ for 10 minutes!
Two step PCR:
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Note:
1) The hot start enzyme used in this product must be activated under pre denaturation conditions of 95 ℃ and 10 minutes.
2) It is recommended to use a two-step PCR reaction program. If good experimental results cannot be obtained due to the use of primers with lower Tm values, a three-step PCR amplification can be attempted.
Three step PCR:
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规格
别名
GoldStar 探针混合物 (UNG)
英文名称
GoldStar Probe Mixture (UNG)
储存条件
-20°C储存,避免反复冻融
运输条件
超低温运输
产品线
生物产品
名称和识别符
分子类型
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