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。
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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
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产品简介
UltraSYBR Mixture（High ROX）是专用于染料法（SYBR Green I）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、SYBR Green I 荧光染料和Mg2+和High ROX校正染料，操作简单方便。主要用于基因组DNA靶序列和RNA反转录后cDNA靶序列的检测。
本品所含的荧光染料SYBR Green I可以与所有的双链DNA结合，使该产品可用于不同靶序列的检测而不需合成特异性标记探针。本品含有的GoldStar Taq DNA Poly- merase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，有效抑制了非特异性的PCR扩增，显著提高了PCR的扩增效率。
所含的ROX染料可校正定量PCR仪孔与孔之间产生的荧光信号误差，本试剂盒中ROX校正染料含量较高，适用于ABI Prism7000/7300/7700/7900，Eppendorf，ABI Step One/Step One Plus等需要较高ROX信号进行校正的荧光定量PCR仪。
产品特点
1．本产品中使用了全新高效热启动酶GoldStar Taq DNA Polymerase与独特的PCR缓 冲体系，显著提高PCR的扩增效率，具有高灵敏度和特异性强的特点。
2．适用于荧光定量PCR检测，能够准确地对目的基因进行定量和检测。
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SYBR Green I荧光染料和ROX染料，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。
4．本品不能用于探针法荧光定量PCR。
5．配制反应液时，请使用新的或者无污染的枪头和离心管，尽量防止污染。
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可得到较好结果，可以终浓度0.1-1.0 μM作为设定范围的参考。
2）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。
3）推荐反应体系为50 μl，也可以根据实际实验需求按比例扩大或者缩小反应体系。
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！
建议采用下表显示的两步法PCR进行程序设定，本程序是以ABI7900荧光定量PCR 仪为例。若因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，三步法操作步骤详见《反应条件的优化》。
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）退火温度请以60-64℃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。
3）本程序是以ABI 7900荧光定量PCR仪为参照设定，融解曲线分析请以所使用的荧光定量PCR 仪推荐的程序进行设定。
反应条件的优化
在荧光定量反应条件优化时，应从引物浓度、退火温度、延伸时间等方面进行考虑， 以提高反应特异性和扩增效率。
1．反应特异性和扩增效率高的实验体系应具备以下条件：
1）反应特异性高：阴性对照无引物二聚体等非特异性扩增；不产生目的片段以外扩增。
2）扩增效率高：Ct值低；PCR扩增效率高，接近理论值100%。 2．反应条件优化方法：
1）引物浓度：通常引物浓度以0.2 μM可得到较好结果，可以终浓度0.1-1.0 μM作为设 定范围的参考。若提高反应特异性，可降低引物浓度；若提高扩增效率，可增加引 物的浓度，由此优化反应体系。
2）退火温度：建议采用两步法PCR，退火温度60℃进行反应。若提高反应特异性，可 提高退火温度，以60-64℃作为设定范围的参考。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，三步法的退火温度请 以56℃-64℃的范围作为设定参考。
3）延伸时间：建议采用两步法PCR，延伸时间1 min进行反应。若提高扩增效率，可尝 试将延伸时间增加，或尝试三步法PCR。
注意！本产品预变性反应必须在95℃ 10分钟下完成！
三步法荧光定量PCR（本程序是以ABI7900荧光定量PCR仪为例）：
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）无法得到理想的扩增效率时，适当降低退火温度；发生非特异性反应时，提高退火温度。
3）若需提高反应扩增效率，可适当增加延伸时间。
4）本程序是以ABI 7900荧光定量PCR仪为参照设定，融解曲线分析请以所使用的荧光定量PCR
仪推荐的程序进行设定。
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Product Introduction
UltraSYBR Mixture (High ROX) is a premixed system dedicated to dye-based (SYBR Green I) real-time fluorescent quantitative PCR at a concentration of 2×, containing GoldStar Taq DNA Polymerase, PCR Buffer, dNTPs, SYBR Green I fluorescent dye and Mg2+ and High ROX correction dyes for easy and convenient operation. It is mainly used for the detection of genomic DNA target sequences and cDNA target sequences after RNA reverse transcription.
The fluorescent dye SYBR Green I contained in this product can bind to all double-stranded DNAs, enabling the product to be used for the detection of different target sequences without the need to synthesize specific labeling probes. GoldStar Taq DNA Poly-merase is a chemically modified, new and highly efficient hot-start enzyme, which 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, and the enzyme activation must be incubated at 95℃ for 10 minutes. The unique combination of PCR buffer system and hot start enzyme effectively inhibits non-specific PCR amplification and significantly improves PCR amplification efficiency.
The ROX dye contained in this kit can correct the fluorescence signal error generated between the wells of the quantitative PCR instrument. This kit contains a high content of ROX correction dye and is suitable for ABI Prism7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus and other fluorescence and quantitative PCR instruments that require a high ROX signal for calibration. quantitative PCR instruments that require a higher ROX signal for correction.
Product Features
1. The new high-efficiency hot-start enzyme GoldStar Taq DNA Polymerase is used in this product with a unique PCR buffer system, which significantly improves the amplification efficiency of PCR with high sensitivity and specificity.
2. Suitable for fluorescent quantitative PCR assays, capable of accurately quantifying and detecting target genes.
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. This product contains SYBR Green I fluorescent dye and ROX dye. Avoid strong light when storing this product or preparing PCR reaction solution.
3. Avoid repeated freezing and thawing of this product. Repeated freezing and thawing may degrade product performance.
4. This product cannot be used for fluorescent quantitative PCR by the probe method.
When preparing reaction solutions, use new or non-contaminated tips and centrifuge tubes to prevent contamination as much as possible.
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
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Note: 1) Usually, a primer concentration of 0.2 μM gives better results, and a final concentration of 0.1-1.0 μM can be used as a reference for setting the range.
2）Usually the amount of DNA template is 10-100ng of genomic DNA or 1-10ng of cDNA as a reference. Since the number of copies of the target gene contained in the templates of different species varies, the templates can be subjected to gradient dilution to determine the optimal amount of template to use.
3）The recommended reaction system is 50 μl, which can be scaled up or down according to the actual experimental requirements.
2. PCR reaction program:
Caution! The pre-denaturation reaction of this product must be completed at 95°C for 10 minutes!
It is recommended to use the two-step PCR shown in the table below to set up the program, and this program is based on the ABI7900 fluorescent quantitative PCR instrument as an example. If you do not get good experimental results due to lower Tm values of primers and other reasons, you can try to carry out three-step PCR amplification, the three-step procedure is detailed in the Optimization of Reaction Conditions.
Note: 1) The hot-start enzyme used in this product shall be activated by the enzyme under the condition of pre-denaturation 95℃ and 10min.
(2) Please use 60-64℃ as the reference for setting range of annealing temperature, and increase the annealing temperature when non-specific reaction occurs.
(3) This program is set up with the ABI 7900 fluorescent quantitative PCR instrument as a reference. For melting curve analysis, please set up the program recommended for the fluorescent quantitative PCR instrument used.
Optimization of reaction conditions
In the optimization of fluorescence quantification reaction conditions, primer concentration, annealing temperature, and extension time should be considered to improve reaction specificity and amplification efficiency.
1. Experimental systems with high reaction specificity and amplification efficiency should have the following conditions:
(1) High reaction specificity: negative control without non-specific amplification such as primer dimer; no amplification beyond the target fragment.
2) High amplification efficiency: low Ct value; high PCR amplification efficiency, close to the theoretical value of 100%.
2. Methods for optimizing reaction conditions:
1) Primer concentration: Usually, a primer concentration of 0.2 μM can get better results, and the final concentration of 0.1-1.0 μM can be used as a reference for setting the range. If you want to improve the specificity of the reaction, you can reduce the primer concentration; if you want to improve the amplification efficiency, you can increase the primer concentration, thus optimizing the reaction system.
2) Annealing temperature: It is recommended to use two-step PCR with an annealing temperature of 60°C for the reaction. If you want to improve the specificity of the reaction, you can increase the annealing temperature, and take 60-64℃ as the reference of the setting range. If you can not get good experimental results due to the use of primers with lower Tm values, etc., you can try to perform three-step PCR amplification, and the annealing temperature of the three-step method, please take the range of 56℃-64℃ as the setting reference.
3) Extension time: it is recommended to use two-step PCR with an extension time of 1min for the reaction. If you want to improve the amplification efficiency, you can try to increase the extension time or try three-step PCR.
Caution! The pre-denaturation reaction of this product must be completed at 95°C for 10 minutes!
Three-step fluorescent quantitative PCR (this program is based on the ABI 7900 fluorescent quantitative PCR instrument):
Note: 1) The hot-start enzyme used in this product shall be activated by the enzyme under the condition of pre-denaturation 95℃ and 10min.
(2) Appropriately reduce the annealing temperature when the desired amplification efficiency cannot be obtained; increase the annealing temperature when a non-specific reaction occurs.
(3) If the reaction amplification efficiency needs to be improved, the extension time can be increased appropriately.
(4) This program is set up with the ABI 7900 Fluorescence Quantitative PCR Instrument as a reference. For melting curve analysis, please set up the program recommended for the fluorescence quantitative PCR instrument used.
规格
别名
UltraSYBR 混合物
规格或纯度
High ROX
英文名称
UltraSYBR Mixture
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
biological_products
名称和识别符
分子类型
小分子
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
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常见问题
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