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，适用于对基线干扰要求严格的色谱和分析工作流程。
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概述
产品内容
2. PCR反应条件
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产品简介
MagicSYBR Mixture是专用于染料法(SYBR GreenⅠ)实时荧光定量PCR的预混体 系，浓度为2×，包含快速Taq DNA Polymerase、PCR Buffer、dNTPs、SYBR Green I荧光染料和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靶序列检测。
本品含有的荧光染料SYBR Green I可以与所有的双链DNA相结合，使用该产品可 用于不同靶序列的检测而不需合成特异性标记探针。含有的快速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30 s，大大缩短了PCR的反应时间。独特的PCR缓冲 体系与热启动酶的组合，有效抑制了非特异产物的产生，显著提高了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. 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。本产品可置于-20℃避光长期 保存。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。
5. 如需更多ROX Reference Dye I，可联系当地业务员。
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。 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）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）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染料的使用量必须与 相应的荧光定量PCR仪相匹配。几种常见仪器的最适ROX Reference Dye I使用量见下表：
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注意：
1）本产品所采用的酶须在预变性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，可将预变性适当时间延长至1-10min，以使起 始模板充分解链，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。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。
3）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，本程序是以Roche 480荧光定 量PCR仪为参照设定。
4）大多数模板可以在40个循环得到很好的扩增曲线，对于低拷贝模板可增加至45个循环内。可根
据实验确定最佳的循环数，以得到更好的扩增曲线。
Products content
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Products Introduction
MagicSYBR Mixture is a pre-mixed system for dye-based (SYBR Green I) real-time fluorescence quantitative PCR. The concentration is 2×, which contains Fast Taq DNA Polymerase, PCR Buffer, dNTPs, SYBR Green I fluorescent dye and Mg2+, which is easy to operate. It is mainly used for the detection of genomic DNA target sequence and cDNA target sequence after RNA reverse transcription.
It contains the fluorescent dye SYBR Green I that binds to all double-stranded DNAs, allowing the detection of different target sequences without the need to synthesize specific labeling probes. The Fast Taq DNA Polymerase can effectively reduce the non-specific amplification caused by 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, which greatly reduces the PCR reaction time. The unique combination of PCR buffer system and hot start enzyme effectively inhibits the generation of non-specific products and significantly improves the amplification efficiency of PCR with 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. This product contains the fluorescent dye SYBR Green I. Avoid strong light when storing this product or preparing PCR reaction solutions.
3. Avoid repeated freezing and thawing of the product, which may degrade its performance. This product can be stored at -20℃ for a long period of time, protected from light. If frequent use is required within a short period of time, store at 2-8°C.
4. This product cannot be used in the probe method of fluorescence quantitative PCR.
5. For more information about ROX Reference Dye I, please contact your local sales representative or call CombiSense customer service.
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.
1. PCR reaction system
Attention:
(1) Usually, a primer concentration of 0.2 μM 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, thus optimizing the reaction system.
(2) Usually, the amount of DNA template is 10-100 ng of genomic DNA or 1-10 ng of cDNA as a reference. Since the copy numbers of target genes contained in the templates of different species are different, the templates can be subjected to gradient dilution to determine the optimal amount of template to be used.
(3) ROX dye is used to correct the fluorescence signal error generated between the wells of the quantitative PCR instrument, and it is generally used in Real Time PCR amplifiers of ABI, Stratagene, etc. The excitation optics of different instruments are different, so the amount of ROX dye used must be matched with the corresponding fluorescence quantitative PCR instrument. The excitation optics vary from instrument to instrument, so the amount of ROX dye used must be matched to the corresponding quantitative fluorescence PCR instrument. The optimal amount of ROX Reference Dye I for several common instruments is shown in the table below:
2.PCR reaction conditions
Attention:
(1) The enzyme used in this product must be activated under the condition of pre-denaturation at 95℃ for 30 s. Most of the templates can be deconvolved well under this condition. Under this condition, most of the templates can be well unchained. For the templates with high GC content and complex secondary structure, the appropriate pre-denaturation time can be extended to 1-10min, so that the starting template can be fully unchained, and the optimal pre-denaturation time can be determined according to the template condition.
(2) It is recommended to use two-step PCR program, the annealing temperature should be 60-64℃ as the reference range, and the annealing temperature can be increased when non-specific reaction occurs. If you can not get good results due to the use of primers with low Tm values, you can try three-step PCR amplification, and the annealing temperature should be set in the range of 56℃-64℃ as a reference.
(3) For melting curve analysis, please set up the program recommended by the fluorescence quantitative PCR instrument you are using, and this program is set up using the Roche 480 fluorescence quantitative PCR instrument as a reference.
4) Most of the templates can get good amplification curves in 40 cycles, which can be increased to 45 cycles for low-copy templates. Can be customized according to
The optimal number of cycles was determined experimentally to get a better amplification profile.
规格
别名
SYBR染料法qPCR mix | MagicSYBR 混合物
英文名称
MagicSYBR Mixture
储存条件
避光,-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
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分子类型
小分子
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