实时聚合酶链反应（real-time PCR），也称为定量聚合酶链反应（qPCR），是一种基于聚合酶链反应（PCR）的流行的分子生物学实验室技术。它可以在PCR期间（即实时）检测目标DNA分子的扩增情况，而不是像常规PCR那样在结束时进行检测。实时PCR可以定量（定量实时PCR）和半定量（即高于或低于一定数量的DNA分子）（半定量实时PCR）使用。实时PCR中有两种检测PCR产物的常用方法，包括（1）结合任何双链DNA的非特异性荧光染料； （2）由寡核苷酸组成的序列特异性DNA探针，所述寡核苷酸用荧光报告物标记，仅在探针与其互补序列杂交后才允许检测。对于第一种方法，对DNA结合染料进行PCR的实时检测有两个要求，即（a）与双链DNA结合时增强荧光；（b）对PCR的抑制作用最小。 SYBR Green主要用于各种qPCR应用中。我们最近开发了新一代的SYBR Green Q4ever Green，用于解决SYBR Green的一些局限性，例如酶抑制。 Q4ever Green可以在PCR中使用，几乎没有PCR抑制作用，并提高了灵敏度。 Q4ever Green可用于检测任何双链DNA序列的扩增。假设您的PCR引物设计合理且反应特性良好，则无需探针，可以减少测定设置和运行成本。作为SYBR Green，主要缺点是它可能产生假阳性信号。即因为Q4ever Green染料与任何双链DNA结合。它也可以与非特异性双链DNA序列结合。设计良好的引物不扩增非靶序列，并进行熔解曲线分析，这一点极为重要。

实验方案

储存在-20°C，避光。 按建议存放时，产品自收到之日起至少稳定12个月。

工作溶液配制

Q4ever Green工作溶液（50X）
使用水或TE缓冲液稀释2000X Q4ever Green储备溶液以制成50X Q4ever Green储备溶液。

操作步骤

建议使用以下协议。 如果需要，可以调整方案以达到最佳效果。

设置PCR反应如下：
5 μL 10X聚合酶缓冲液（不含镁）
2.5 μL的50 mM MgCl₂
2 μL 50X Q4ever Green工作溶液
2 μL的5 mM dUTP
1-5个单位的DNA聚合酶
加入所需的cDNA
每个引物100-1000 nM（正向和反向引物的终浓度）
用dH₂O将最终体积调整为50 μL。
在热循环荧光计上执行实时PCR并记录荧光信号。

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