检测增强剂,用于实时 PCR
检测增强剂,用于实时 PCR
Applied Biosystems™

检测增强剂,用于实时 PCR

检测增强剂配方独特,可改善实时 PCR (qPCR) 工作流程中的模板扩增。此试剂特别适用于含有富含 GC 序列和/或稳定二级结构的 DNA 样本。检测增强剂是了解更多信息
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货号反应次数
A448111000 × 25 μL 反应
A44941500 × 25 μL 反应
A44810100 × 25 μL 反应
货号 A44811
价格(CNY)
781.00
Each
添加至购物车
反应次数:
1000 × 25 μL 反应
价格(CNY)
781.00
Each
添加至购物车
检测增强剂配方独特,可改善实时 PCR (qPCR) 工作流程中的模板扩增。此试剂特别适用于含有富含 GC 序列和/或稳定二级结构的 DNA 样本。检测增强剂是 AgPath-ID 一步法 RT-qPCR 试剂盒的一个组分,在此作为独立产品提供,可与任何 Applied Biosystem 实时 PCR 预混液配套使用。

•改善包含高 GC 含量和反复出现二级结构的模板的 qPCR 扩增
• 可兼容任何 Applied Biosystem 实时荧光定量 PCR 预混液
• 灵活的试剂盒规格适用于各种实验室通量

高 GC 含量样品
高 GC 含量的 DNA 序列通常被定义为含有>60%胞嘧啶 (C) 或鸟嘌呤 (G) 的 DNA 序列。富含 GC 的样本扩增可能特别具有挑战性,因为其序列比 GC 含量较低的样本更稳定。在 qPCR 实验中,此现象会导致更高的 DNA 熔点。此外,富含 GC 的序列可形成二级结构,这会进一步增加扩增的挑战。在反应混合物中加入少量检测增强剂可以显著改善富含 GC 样本的 qPCR 扩增。

增强扩增
检测增强剂是专门配制的,用于解决特别稳定的 DNA 序列的 qPCR 扩增问题。仅适用于扩增受到富含 GC 序列和/或含有稳定二级结构的模板影响的实时 PCR 工作流程。此试剂可能会影响其它靶标类型的灵敏度,因此建议首先在不使用检测增强剂的情况下构建反应。如果从预期为阳性的样品中未检测到信号,再向反应中加入检测增强剂。

每 25 µL 反应体系中推荐加入的检测增强剂体积为 1.67 µL。有关检测增强剂应用的更多信息,请参见 AgPath-ID 一步法 RT-qPCR 预混液产品说明书
仅供科研使用。不可用于诊断程序。
规格
适用于(设备)7500 系统
反应次数1000 × 25 μL 反应
产品线Applied Biosystems
产品类型检测增强剂
数量1000 reactions
样品类型病毒样本
运输条件干冰
容量2.4 mL
检测方法引物-探针
适用于(应用)实时 PCR (qPCR)
高 GC PCR 扩增效果
PCR 方法一步法 RT-qPCR
反应速度标准
Unit SizeEach
内容与储存
2 x 1.2 mL 检测增强剂,保存于 -5 至 -30°C 下

常见问题解答 (FAQ)

With the AgPath-ID One-Step RT-PCR Reagents, I am getting signal detection in the no-template control (NTC) reaction. Why is this?

This is likely due to PCR contamination. Here are some recommendations:

- Repeat the qRT-PCR reaction with fresh reagents and decontaminated pipettors.
- Set up and run the qRT-PCR reaction in an area that is isolated from areas used for nucleic acid isolation and PCR product analysis.
- The Reverse Transcriptase enzyme contained in this kit is produced using an E. coli expression vector containing a proprietary version of the MMLV pol gene (GenBank accession no. J02255) expressed from pET-24(+). It is possible that a minimal amount of the expression vector could be carried over into the final mastermix formulation. If you are targeting MMLV, a related virus, or any of the plasmid sequence, we recommend designing primer sequences for target sequences not contained in the expression vector.

What can I do to improve the sensitivity of my qPCR assay?

If you are targeting a low-abundance gene, you may have trouble getting Ct values in a good, reliable range (Ct > 32). To increase the sensitivity of the assay, you may want to consider the following:

- Increase the amount of RNA input into your reverse transcription reaction, if possible
- Increase the amount of cDNA in your qPCR reaction (20% by volume max)
- Try a different reverse transcription kit, such as our SuperScript VILO Master Mix, for the highest cDNA yield possible
- Consider trying a one-step or Cells-to-CT type workflow (depending on your sample type)

How do I set the baseline for my qPCR experiment?

Most times your instrument software can automatically set a proper baseline for your data. Check out our short video, Understanding Baselines, for more information on how to set them (https://www.youtube.com/watch?feature=player_embedded&v=5BjFAJHW-bE).

How do I set the threshold for my qPCR experiment?

In most cases your instrument software can automatically set a proper threshold for your data. Check out our short video, Understanding Thresholds, for more information on how to set them (https://www.youtube.com/watch?feature=player_embedded&v=H_xsuRQIM9M).

I am not getting any amplification with my TaqMan Assay or SYBR Green primer set. What is causing this?

There could be several reasons for no amplification from an assay or primer set. Please see these examples and suggested solutions in our Real-Time Troubleshooting Tool (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/no-amplification.html) for more details.