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
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
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
本产品是一种检测人类男性Y染色体浓度的实时荧光定量PCR试剂盒，包括精心优 化的PCR反应液、引物混合物、标准品，尤其适用于珍贵、微量DNA样本的定量检测。 试剂盒采用了全新高效、快速的热启动扩增酶GoldStar Taq DNA Polymerase，有效避免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。本品实现对Y染色体的准确定量，后续可应用于各种领域如遗传制图、物种多态性研究、疾病基因定位、亲子鉴定和法医鉴定等研究分析。
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3中说明。
适用范围
本产品适用于科研、临床、法医学和亲子鉴定等领域对男性Y染色体DNA的定量检测。
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0.05-10 ng/μL之间。4℃冰上放置备用。
2. 标准品稀释：按照下表，先将Human DNA Standard（100ng/uL）用TE按下表稀释出5个不同浓度的标准品。10 ng/μL的DNA Standard 1（Std. 1）可在-20℃稳定保存1个月；Std2-5只能当天使用，配好后暂时不用时应4℃或冰上放置。
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3. qPCR反应体系配制
配制前预先将所需要用到的冷冻保存的试剂完全融化并多次颠倒混匀，然后短暂离心后备用。标准品和模板按照上面所述的方法稀释后备用。
20 μL 的基础反应体系如下：
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注意： High ROX机型：每50μL反应体系加入1μL 50 ×High ROX；
Low ROX机型：每500 μL反应体系加入1 μL50 ×High ROX。
根据需要配出足够量的反应体系混合物，反应体系配完并充分混匀后，按每孔16 μl 体积加入反应孔中。然后将准备好的标准品及稀释好的样品加入对应反应孔，加入量为4 μL /孔。空白对照管中加入TE，同样加入量为4 μL /孔。
推荐使用20 μL 反应，如需进行更小体系反应，将体系各组分等比减少即可。
4. qPCR反应程序
本试剂盒的PCR mix 含目的基因的FAM荧光探针和内参照Internal PCR Control (IPC)的VIC荧光探针， 检测时需要选择水解探针双荧光的qPCR程序。请根据所用仪器说明进行设定。PCR反应温度条件如下
1. 标准曲线制作
参照数据处理Excel表绘制标准曲线。标准曲线相关系数R2应不低于0.98，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。
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2. 结果分析及浓度计算
目的基因FAM信号的实验复孔间的Ct差异应不超过0.3，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计算样品的浓度。
具体计算方法请参见本产品的数据处理Excel。
若FAM信号不正常，需分析内参照Internal PCR Control (IPC)的VIC信号，确认PCR反应过程是否异常。若样品空VIC的Ct值显著大于标准品或空白对照孔，说明样品对PCR反应有抑制。
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 content
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Product Introduction
This product is a real-time fluorescence quantitative PCR kit for detecting the concentration of human male Y chromosome, including carefully optimized PCR reaction solution, primer mixture and standards, especially suitable for the quantitative detection of precious and micro DNA samples. The kit adopts a new efficient and fast hot-start amplification enzyme GoldStar Taq DNA Polymerase, which effectively avoids non-specific amplification caused by non-specific binding of primers and templates or primer dimerization at room temperature. This product realizes accurate quantification of Y chromosome and can be applied in various fields such as genetic mapping, species polymorphism research, disease gene localization, paternity testing and forensic analysis.
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, ViiA 7 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 3.
Scope of application
This product is suitable for quantitative testing of male Y chromosome DNA in scientific research, clinical, forensic medicine and paternity testing.
Usage
1. 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 0.05-10 ng/μL. 4°C on ice was set aside.
2. Standard dilution: according to the following table, firstly dilute Human DNA Standard (100ng/uL) with TE to make 5 standards of different concentrations according to the table below. 10ng/μL of DNA Standard 1 (Std.1) can be stored stably at -20℃ for 1 month; Std2-5 can only be used on the same day, and should be placed at 4℃ or on ice when not in use for the time being after preparation. When Std2-5 are not used temporarily after preparation, they should be stored at 4℃ or on ice.
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3. qPCR reaction system preparation
Before preparation, the cryopreserved reagents to be used were completely melted and mixed by inverting several times, then centrifuged briefly and prepared. Standards and templates were diluted as described above and prepared.
The base reaction system for 20 μL was as follows:
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Note: High ROX model: add 1 μL of 50×High ROX per 50 μL of reaction system; Low ROX model: add 1 μL of 50×High ROX per 500 μL of reaction system.
A sufficient amount of reaction system mixture was prepared according to the need, and after the reaction system was prepared and mixed thoroughly, it was added to the reaction wells in a volume of 16 μl per well. Then add the prepared standards and diluted samples into the corresponding reaction wells, the amount of addition is 4μL/well. TE was added to the blank control tube, and the same amount was added at 4 μL/well.
It is recommended to use 20 μL for the reaction, if you need to perform a smaller system reaction, reduce the system components in equal proportion.
4. qPCR reaction program
The PCR mix of this kit contains a FAM fluorescent probe for the target gene and a VIC fluorescent probe with internal reference to Internal PCR Control (IPC). qPCR program with dual fluorescence of hydrolyzed probes needs to be selected for the assay. Please follow the instructions of the instrument used to set up the qPCR program, and the PCR temperature conditions are as follows:
1. Standard curve production
The standard curve was plotted with reference to the Excel sheet for data processing. The correlation coefficient R2 of the standard curve should be not less than 0.98, and the slope should be located between -3.1 and -3.6 when the Ct value is used as the longitudinal coordinate. If the parameters of the standard curve are unreasonable, it is recommended to repeat the experiment.
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2. Analysis of results and calculation of concentrations
The Ct difference between experimental replicate wells for FAM signaling of the target gene should be no more than 0.3, otherwise invalid data need to be deleted or the experiment needs to be repeated, do not use Ct outside the valid Ct range of the standard curve to calculate the concentration of the sample.
For specific calculations, please refer to the data processing Excel for this product.
If the FAM signal is abnormal, the VIC signal of the internal reference Internal PCR Control (IPC) needs to be analyzed to confirm whether the PCR reaction process is abnormal. If the Ct value of the sample null VIC is significantly larger than that of the standard or blank control wells, it means that the sample inhibits the PCR reaction.
matters needing attention
1. Before testing, these instructions should be read in detail. It should be operated by personnel with professional experience or qualified by training.
2. For use, please mix gently by turning up and down, avoid foaming as much as possible, and use it after centrifugation for a short period of time.
3. Avoid repeated freezing and thawing of the product, repeated freezing and thawing may degrade the performance of the product.
4. When preparing the reaction solution, please use new or non-contaminated tips and centrifuge tubes to prevent contamination as much as possible.
规格
英文名称
Y Human Male DNA Quantification Kit
储存与运输
储存条件
-20°C储存,避免反复冻融
运输条件
超低温运输
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
C of A & Other Certificates(BSE/TSE, COO):
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技术文档和文章
溶液计算器
产品仅提供给经过验证的企业、机构和合格的专业人员用于研发。不适用于人类、动物、诊断或治疗。
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