PCR Series - Chemical Dye Method Quantitative PCR Premix (Independent ROX)
PCR Series - Chemical Dye Method Quantitative PCR Premix (Independent ROX)

PCR Series - Chemical Dye Method Quantitative PCR Premix (Independent ROX)

Taq HS qPCR Premium is SYBR ® Green I chimeric dye method specific qPCR reagent, 2 × Pre mixed solution, containing all qPCR components except primers and DNA samples, can reduce operational steps, shorten sample addition time, and reduce the probability of contamination.

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Article NumberProduct NameChinese NameSpecificationsCatalog Price (yuan)
DQ00101Taq-HS qPCR Premix (Universal)Quantitative PCR premix using chemical dye method (general)2 ×，4 ml688  

Taq HS qPCR Premium is SYBR ® Green I chimeric dye method specific qPCR reagent, 2 × Pre mixed solution, containing all qPCR components except primers and DNA samples, can reduce operational steps, shorten sample addition time, and reduce the probability of contamination. Its core component is the chemically modified hot start Taq DNA polymerase, combined with the carefully optimized Buffer system, which can effectively inhibit non-specific amplification, accurately quantify templates in a wide concentration range, and obtain stable and reliable qPCR results.


The dye method is cost-effective and can perform dissolution curves to analyze the TM values of all PCR products

Problem Description


                     Possible Causes

 


                                                                Solution

 

The amplification curve is not smooth


  The fluorescence signal is too weak and generated after system correction

 

Ensure that the pre mixed dyes in Mix are not degraded; Replacing fluorescent signal collection with better qPCR specific consumables

Amplification curve breaks or slides


The template concentration is high, and the baseline endpoint value is greater than the Cq value

 

Reduce the baseline endpoint (Cq value -4) and reanalyze the data

Sudden drop in amplification curve of individual wells

There are bubbles left in the reaction tube

Ensure that Mix is completely dissolved, do not vortex shake and mix well

After adding samples, gently flick and centrifuge to remove bubbles

Extend the pre denaturation time to 10 minutes to remove bubbles

反应结束无扩增曲线出现

反应循环数偏少

设置循环数为40，但更多的循环数会增加过多的背景信号

荧光信号采集步骤未设置或者设置错误

两步法扩增程序一般将信号采集设置在退火&延伸阶段，三步法扩增程序应当将信号采集设置在72℃延伸阶段

引物可能降解

长期未用的引物，应先通过PAGE电泳检测完整性，以排除其降解的可能

模板浓度过低

减少模板稀释倍数重复实验，样品浓度未知的情况下先从最高浓度做起

模板降解

 

重新制备模板，重复实验

Cq值出现过晚

扩增效率低

提高引物浓度，尝试三步法扩增程序，或者重新设计引物

模板浓度过低

减少模板稀释倍数重复实验，样品浓度未知的情况下先从最高浓度做起

模板降解

 

重新制备模板，重复实验

扩增产物过长 

扩增产物长度控制在 80~200 bp

体系中存在 PCR 抑制剂

一般为模板带入，加大模板稀释倍数或重新制备纯度高的模板重复实验

空白对照出现信号

反应体系污染

首先更换空白对照的水，如果还发生同样情况，继续更换引物、吸头、PCR 管或启用新的 Mix

反应体系在超净工作台内配制，减少气溶胶污染

出现引物二聚体等非特异性扩增

一般在35 循环以后空白对照出现扩增产物属正常情况，应配合熔解曲线进行分析，重新设计引物，调整引物浓度或优化 PCR 反应程序

熔解曲线出现多峰


                     引物设计不佳

 

根据引物设计原则重新设计新引物


                     引物浓度过高

 

适当降低引物浓度

cDNA 模板存在基因组污染

提取后的 RNA 溶液使用 DNA 酶进行消化，例如：dsDNase，以去除基因组 污染，或设计跨内含子引物

RNA 溶液使用 DNA 酶进行消化，例如：dsDNase，以去除基因组

污染，或设计跨内含子引物

实验重复性差

加样误差大

使用精准的移液器、配合高品质吸头准确移液

高倍稀释模板，加入大体积模板减少加样误差

放大 qPCR 反应体积


                    模板浓度过低

 

减少模板稀释倍数重复实验


          qPCR 仪不同位置的温度偏差

 

定期校准 qPCR 仪






Quantitative PCR...

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