| 货号 (SKU) | 包装规格 | 是否现货 | 价格 | 数量 |
|---|---|---|---|---|
| N665917-1ml |
1ml |
期货  |
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| N665917-5ml |
5ml |
期货 |
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| 规格或纯度 | for Illumina | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| 英文名称 | NGS cfDNA Library Quantification Kit | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 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 |
产品简介 本产品是针对cfDNA的染料法（SYBR Green I）qPCR NGS文库定量试剂盒，提供了qPCR过程所需的反应混合液，DNA引物混合物、标准品以及样品稀释液，试剂体系完整，操作简单方便。反应混合物中所含的荧光染料SYBR Green I 可以与所有的双链DNA结合。本试剂盒使用的是一种经化学修饰的全新高效热启动聚合酶，酶的激活需在95℃下孵育10 min。该产品特异性强、扩增效率高，试剂盒中的标准品长度（约270 bp） 与cfDNA NGS文库的平均长度（250-300 bp）相当，能够对构建的cfDNA文库浓度进行快速准确的定量。 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： Roche LightCycler 480，Roche LightCyler 96，Bio-rad iCyler iQ，iQ5，CFX96等。 需要Low ROX校正的仪器： 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： ABI Prism7000/7300/7700/7900，Eppendorf，ABI Step One/Step One Plus等。 注：High Rox和Low Rox 的配制方法见使用方法2中说明。 适用范围 本产品是针对Illumina平台二代测序文库浓度绝对定量而设计。文库末端包含Illumina P5和P7芯片结合序列，长度不超过1 kb，浓度不低于0.02 pM即可使用本品进行定量实验。试剂盒提供的qPCR Primer Mix中包含如下两种引物序列： Primer 1: 5'-AAT GAT ACG GCG ACC ACC GA-3' Primer 2: 5'-CAA GCA GAA GAC GGC ATA CGA-3' 可预先通过引物序列确认文库是否可以被该引物对扩增。 使用方法 1. 扩增模板准备 将待检测文库样品用TE（10 mM Tris-Cl，pH8.0，1mM EDTA）稀释，稀释后浓度尽量在0.01-60 pM之间。4℃冰上放置备用。 2. qPCR反应体系配制 配制前预先将所需的冷冻保存试剂完全融化并多次颠倒混匀，然后短暂离心后备用。 20 μl的基础反应体系如下：
说明： High Rox机型：每50 μl反应体系加入1 μl High Rox； Low Rox机型：每500 μl反应体系加入1 μl High Rox。 根据需要配出足够量的反应体系混合物，混匀后按每孔16 μl体积加入至反应孔中，空白对照加入同样体积的TE，再将准备好的标准品和稀释的样品加入至对应反应孔中， 加入量为4 μl/孔。推荐使用20 μl反应体系，如需进行更小体系反应，将体系各组分等比减少即可。 3. qPCR反应程序
1） 如文库平均长度大于700 bp，应适量增加退火/延伸时间。 2） 溶解曲线参照具体仪器设定程序。 数据分析 1. 标准曲线制作 照数据处理Excel表绘制标准曲线。标准曲线相关系数R2应不低于0.99，以Ct值为纵 坐标时，斜率应位于-3.1与-3.6之间，如标准曲线参数不合理，建议重复实验。
2. 文库浓度计算 实验三个复孔间的Ct差异应不超过0.2，否则需删除无效数据或重复实验，请勿使用 标准曲线有效Ct范围外的Ct计算稀释文库的浓度。具体文库浓度计算方法请参见本 产品的数据处理Excel。 注意事项 1. 在试验前，应详细阅读本说明。应由具备专业经验或经培训合格的人员进行操作。 2. 使用请上下颠倒轻轻混匀，尽量避免起泡，并经短暂离心后使用。 3. 避免反复冻融本品，反复冻融可能使产品性能下降。 4. 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。
Product Introduction This is a dye-based (SYBR Green I) qPCR NGS library quantification kit for cfDNA, which provides the reaction mixture, DNA primer mixture, standards, and sample dilutions required for the qPCR process, making it a complete reagent system that is easy and convenient to use. The fluorescent dye SYBR Green I contained in the reaction mixture binds to all double-stranded DNA. The kit uses a new chemically modified high-efficiency hot-start polymerase, the activation of the enzyme needs to be incubated at 95 ℃ for 10 min. the product is highly specific, high amplification efficiency, the length of the standard in the kit (about 270bp) is comparable to the average length of the cfDNA NGS libraries (250-300bp), which is able to quickly and accurately quantitate the concentration of the constructed cfDNA libraries. quantification. 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: Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96, etc. Instruments requiring Low ROX calibration: ABI Prism7500/7500 Fast, QuantStudio®3 System, QuantStudio®5 System, QuantStudio®6 Flex System, QuantStudio®7 Flex System, ViiA7 System, Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, and others. Instruments requiring High ROX calibration: ABI Prism7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, etc. NOTE: High Rox and Low Rox are formulated as described in Method of Use 2. Applicable scope This product is designed for the absolute quantification of the concentration of Illumina platform second generation sequencing libraries. The end of the library contains Illumina P5 and P7 microarray binding sequences, the length of which does not exceed 1kb, and the concentration is not less than 0.02pM can be used for quantitative experiments. The qPCR Primer Mix provided in the kit contains the following two primer sequences: Primer 1:5'-AAT GAT ACG GCG ACC ACC GA-3' Primer 2: 5'-CAA GCA GAA GAC GGC ATA CGA-3' The primer sequence can be used in advance to confirm whether the library can be amplified by that primer pair. Usage Amplification template preparation The library samples to be detected were diluted with TE (10 mM Tris-Cl, pH 8.0, 1 mM EDTA), and the concentration after dilution was as close as possible to the range of 0.01-60 pM. 4°C on ice was set aside. qPCR reaction system preparation The desired cryopreservation reagent is pre-melted completely and mixed by inverting several times before preparation, then centrifuged briefly and set aside. The base reaction system for 20 μl was as follows:
Description: High Rox model: 1 μl High Rox per 50 μl of reaction system; Low Rox model: 1 μl High Rox per 500 μl of reaction system. Prepare a sufficient amount of reaction system mixture according to the need, mix well and add to the reaction wells in a volume of 16 μl per well, add the same volume of TE to the blank control, and then add the prepared standards and diluted samples to the corresponding reaction wells in a volume of 4 μl/well. It is recommended to use 20 μl reaction system, if you need to carry out a smaller system reaction, the system components can be reduced in equal proportion. 3.qPCR reaction program
If the average length of the library is greater than 700bp, the annealing/extension time should be increased appropriately. Refer to the specific instrument setup program for dissolution curves. data analysis Standard curve production The standard curve was plotted according to the data processing Excel sheet. The correlation coefficient R2 of the standard curve should be not less than 0.99, and the slope should be located between -3.1 and -3.6 when the Ct value is the longitudinal coordinate. If the parameters of the standard curve are unreasonable, it is recommended to repeat the experiment.
Library Concentration Calculations The difference in Ct between the three replicate wells of the experiment should be no more than 0.2, otherwise the invalid data should be deleted or the experiment should be repeated. Do not use the Ct outside the valid Ct range of the standard curve to calculate the concentration of the diluted libraries. Please refer to the data processing Excel of this product for the specific library concentration calculation method. matters needing attention These instructions should be read in detail before testing. It should be carried out by personnel with specialized experience or qualified by training. Mix gently by turning up and down, avoid foaming as much as possible, and centrifuge for a short time before use. Avoid repeated freezing and thawing of this product; repeated freezing and thawing may degrade product performance. When preparing reaction solutions, use new or non-contaminated tips and centrifuge tubes to prevent contamination as much as possible |
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