基本描述

规格或纯度

for Ion torrent

英文名称

NGS 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

N665925	Component	1 mL	5 mL	Storage
N665925A	2×SYBR qPCR Master Mix 1	1 mL	5 mL	-20℃. Avoid freeze/ Thaw cycle.
N665925B	qPCR Primer Mix 1	100 μL	500 μL	-20℃. Avoid freeze/ Thaw cycle.
N665925C	DNA Standard I	100 μL	500 μL	-20℃. Avoid freeze/ Thaw cycle.
N665925D	DNA Standard II	100 μL	500 μL	-20℃. Avoid freeze/ Thaw cycle.
N665925E	DNA Standard III	100 μL	500 μL	-20℃. Avoid freeze/ Thaw cycle.
N665925F	DNA Standard IV	100 μL	500 μL	-20℃. Avoid freeze/ Thaw cycle.
N665925G	DNA Standard V	100 μL	500 μL	-20℃. Avoid freeze/ Thaw cycle.
N665925H	50×High ROX	40 μL	200 μL	-20℃. Avoid freeze/ Thaw cycle.

产品简介

本产品是采用染料法（SYBR Green I）对NGS建库后的产物进行实时荧光定量PCR

（qPCR）。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；使用的GoldStar Taq DNA Polymerase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，能够对构建的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Ion torrent平台二代测序文库浓度绝对定量而设计。文库末端包含Ion torrent P5和P7芯片结合序列，长度不超过1kb，浓度不低于0.005pM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'-CCA TCT CAT CCC TGC GTG TC - 3'

Primer 2: 5'-CCT CTC TAT GGG CAG TCG GTG AT-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0.05-50 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：

试剂	20 μl反应体系
2×SYBR qPCR MasterMix	10 μl
qPCR Primer Mix 1	0.8 μl
Template	4 μl
ddH₂O	5.2 μl

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）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。

2）如文库平均长度大于700 bp，应适量增加退火/延伸时间。

数据分析

1. 标准曲线制作

使用有效范围内的Ct值绘制标准曲线。标准曲线相关系数R2应不低于0.99，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
DNA Standard I	50 pM
DNA Standard II	5 pM
DNA Standard III	0.5 pM
DNA Standard IV	0.05 pM
DNA Standard V	0.005 pM

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。

N665925	Component	1 mL	5 mL	Storage
N665925A	2×SYBR qPCR Master Mix 1	1 mL	5 mL	-20℃. Avoid freeze/ Thaw cycle.
N665925B	qPCR Primer Mix 1	100 μL	500 μL	-20℃. Avoid freeze/ Thaw cycle.
N665925C	DNA Standard I	100 μL	500 μL	-20℃. Avoid freeze/ Thaw cycle.
N665925D	DNA Standard II	100 μL	500 μL	-20℃. Avoid freeze/ Thaw cycle.
N665925E	DNA Standard III	100 μL	500 μL	-20℃. Avoid freeze/ Thaw cycle.
N665925F	DNA Standard IV	100 μL	500 μL	-20℃. Avoid freeze/ Thaw cycle.
N665925G	DNA Standard V	100 μL	500 μL	-20℃. Avoid freeze/ Thaw cycle.
N665925H	50×High ROX	40 μL	200 μL	-20℃. Avoid freeze/ Thaw cycle.

Product Introduction

This product is a real-time fluorescence quantitative PCR of the products after NGS library construction using a dye method (SYBR Green I).

(qPCR). The kit provides the reaction mixes, DNA primer mixtures, standards and sample dilutions required for the qPCR process, making the reagent system complete and easy to use. The fluorescent dye SYBR Green I contained in the reaction mixture can bind to all double-stranded DNA; the GoldStar Taq DNA Polymerase used is a chemically modified new high-efficiency hot-start polymerase, and the activation of the enzyme needs to be incubated at 95℃ for 10 minutes. the product has high specificity, high amplification efficiency, and is able to quickly and accurately quantify the concentration of the constructed libraries. The product is highly specific and efficient in amplification, and can quickly and accurately quantify the concentration of the constructed library.

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 Use 2.

Scope of application

This product is designed for absolute quantification of the concentration of Ion torrent platform second generation sequencing libraries. The end of the library contains Ion torrent P5 and P7 microarray binding sequences, the length of which does not exceed 1kb, and the concentration is not less than 0.005pM can be used to perform quantitative experiments with this product. The qPCR Primer Mix provided in the kit contains the following two primer sequences:

Primer 1:5'-CCA TCT CAT CCC TGC GTG TC - 3' Primer 2: 5'-CCT CTC TAT GGG CAG TCG GTG AT-3'

The primer sequence can be used in advance to confirm whether the library can be amplified by that primer pair.

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 0.05-50 pM. 4°C on ice was set aside.

2. 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:

Reagent	20 μl Reaction system
2×SYBR qPCR MasterMix	10 μl
qPCR Primer Mix 1	0.8 μl
Template	4 μl
ddH₂O	5.2 μl

Description: High Rox model: add 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

1) Please use 60-64℃ as a reference for setting range of annealing temperature, and increase the annealing temperature when non-specific reaction occurs.

2) If the average length of the library is greater than 700bp, the annealing/extension time should be increased appropriately.

data analysis

1. Standard curve production

The standard curve was plotted using Ct values in the valid range. The standard curve correlation coefficient R2 should not be less than 0.99 and the slope should lie between -3.1 and -3.6. If the standard curve parameters are not reasonable, it is recommended to repeat the experiment.

DNA Standard Name	DNA Standard Concentration
DNA Standard I	50 pM
DNA Standard II	5 pM
DNA Standard III	0.5 pM
DNA Standard IV	0.05 pM
DNA Standard V	0.005 pM

2. Library concentration calculation

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

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.

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

n665925_ngs library quantification kit for ion torrent.pdf

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货号 (SKU)	包装规格	是否现货	价格	数量
N665925-1ml	1ml	期货
N665925-5ml	5ml	期货

NGS文库定量试剂盒

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