基本描述

英文名称

Human Genomic DNA 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

产品内容

H665940	Component	1 mL	5 mL	Storage
H665940A	2×GoldStar TaqMan Mixture	1 mL	5×1 mL	-20°C. Avoid freeze/thaw cycle.
H665940B	Primer Mix 1	300 µL	5×300 µL	-20°C. Avoid freeze/thaw cycle.
H665940C	Human DNA Standard 1 (100 ng/µL)	100 µL	5×100 µL	-20°C. Avoid freeze/thaw cycle.
H665940D	50×High rox	40 µL	200 µL	-20°C. Avoid freeze/thaw cycle.

产品简介

本产品是采用探针法进行实时荧光定量PCR（qPCR），实现对从各种样品（石蜡样本、流式分选的细胞、血清或血浆样本及少量临床样本等）中抽提的DNA进行浓度和质量的准确检测。产品提供了qPCR过程中所需的全套试剂，包括反应混合液，引物混合物，标准品，只需添加提取好的DNA即可开始实验，操作简单方便、省时省力。反应混合物使用了高效、快速的热启动GoldStar Taq DNA Polymerase，扩增灵敏度高，特异性好，缩短了程序反应的时间。

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人基因组DNA样品浓度的定量检测。

使用方法

1. 扩增模板准备

将待检测样品用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 1（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。

标样	对应浓度(ng/μl)	最小稀释体积（单位：μl）
Std.1	10	10 [100 ng/μl DNA Standard 1]+ 90 TE
Std.2	2.5	20 [Std. 1] +60 TE
Std.3	0.625	20 [Std. 2] +60 TE
Std.4	0.15625	20 [Std. 3] +60 TE
Std.5	0.0390625	20 [Std. 4] +60 TE

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。20 μl的基础反应体系如下。

20 μl的基础反应体系如下：

试剂	20 µI反应体系
2×GoldStar TaqMan Mixture	10 μl
Primer Mix	3 μl
Template	4 μl
ddH₂O	3 μl

注意： High Rox机型：每50 μl反应体系加入1 μl 50×High Rox；

Low Rox机型：每500 μl反应体系加入1 μl 50×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：

数据分析

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。

DNA Standard名称	DNA Standard浓度（ng/μl）
DNA Standard 1	10
DNA Standard 2	2.5
DNA Standard 3	0.625
DNA Standard 4	0.15625
DNA Standard 5	0.0390625

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。

样品浓度的具体计算参照数据处理Excel表。

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。若样品孔VIC信号的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

H665940	Component	1 mL	5 mL	Storage
H665940A	2×GoldStar TaqMan Mixture	1 mL	5×1 mL	-20°C. Avoid freeze/thaw cycle.
H665940B	Primer Mix 1	300 µL	5×300 µL	-20°C. Avoid freeze/thaw cycle.
H665940C	Human DNA Standard 1 (100 ng/µL)	100 µL	5×100 µL	-20°C. Avoid freeze/thaw cycle.
H665940D	50×High rox	40 µL	200 µL	-20°C. Avoid freeze/thaw cycle.

Product Introduction

This product is a real-time fluorescence quantitative PCR (qPCR) using the probe method, which realizes the accurate detection of the concentration and quality of DNA extracted from various samples (paraffin samples, flow-sorted cells, serum or plasma samples and a small number of clinical samples, etc.). The product provides a full set of reagents required in the qPCR process, including reaction mix, primer mixtures, and standards. Simply add the extracted DNA and start the experiment, which is easy and convenient to operate and saves time and labor. The reaction mixture uses highly efficient and fast hot-start GoldStar Taq DNA Polymerase, which has high amplification sensitivity, good specificity and shortens the program reaction time.

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.

Applicable scope

This product is suitable for quantitative detection of human genomic DNA sample concentration in scientific research, clinical, forensic medicine and paternity testing.

Usage

1. Amplification template preparation

The 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. The samples were placed on ice at 4°C and set aside.

2. Standard dilution: according to the following table, firstly dilute Human DNA Standard 1 (100ng/ul) with TE to make 5 different concentrations of standard 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 not used temporarily after preparation, it should be kept at 4℃ or on ice.

Standard sample	Corresponding concentration(ng/μl)	Minimum Dilution Volume (Unit: μ L)
Std.1	10	10 [100 ng/μl DNA Standard 1]+ 90 TE
Std.2	2.5	20 [Std. 1] +60 TE
Std.3	0.625	20 [Std. 2] +60 TE
Std.4	0.15625	20 [Std. 3] +60 TE
Std.5	0.0390625	20 [Std. 4] +60 TE

3. qPCR reaction system preparation

The cryopreserved reagents to be used were completely melted and mixed by inversion several times before preparation, and then briefly centrifuged and prepared for use. 20 μl of the base reaction system was as follows.

The base reaction system for 20 μl was as follows:

Reagent	20 μl Reaction system
2×GoldStar TaqMan Mixture	10 μl
Primer Mix	3 μl
Template	4 μl
ddH₂O	3 μl

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 added 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 of reaction, if you need to carry out a smaller system reaction, the system components can be reduced in equal proportion.

4. qPCR reaction program

The PCR mix in 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 set up the program according to the instructions of your instrument.

PCR reaction temperature conditions were as follows:

Data analysis
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.

DNA Standard Name	DNA Standard Concentration（ng/μl）
DNA Standard 1	10
DNA Standard 2	2.5
DNA Standard 3	0.625
DNA Standard 4	0.15625
DNA Standard 5	0.0390625

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.

Specific calculations of sample concentrations were made with reference to the data processing Excel sheet.

If the FAM signal is abnormal, the VIC signal of the internal reference Internal PCR Control (IPC) should be analyzed to confirm whether the PCR reaction process is abnormal. If the Ct value of the VIC signal of the sample wells is significantly larger than that of the standard or blank control wells, it means that the sample has inhibited 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 try to prevent contamination.

质检证书（CoA,COO,BSE/TSE 和分析图谱)

C of A & Other Certificates(BSE/TSE, COO):

输入批号以搜索分析图谱:

技术文档和文章

h665940_human genomic dna quantification kit.pdf

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货号 (SKU)	包装规格	是否现货	价格	数量
H665940-1ml	1ml	期货
H665940-5ml	5ml	期货

人类基因组DNA定量试剂盒

库存信息