RTGreen是一款用于实时荧光定量PCR染料，也是一种新型的专门为 qPCR和高分辨率熔解曲线分析设计的DNA结合染料，它通过一种 “按需求释放” 的机制，极大地降低了对 PCR 扩增的抑制性。该染料的诸多优点使它远胜于 SYBR Green l。除了有相似的光谱特性，RTGreen有三个主要特点使它区别于 SYBR GreenI:
1) 对 PCR的抑制性远小于 SYBR Green l，进行的qPCR实验可以使用快速 PCR步骤。
2) 在实验中可以使用较高的浓度，从而获得远强于SVBR Green l 扩增信号。较高浓度的RTGreen也消除了 “染料重分布” 的缺陷，使RTGreen既可用于多重 PCR，也可用于高分辦率 (高清晰) 溶解曲线分析 (HRM)。
3) 该分析正被越来越多的用于PCR后的基因分型和异源双链分析。
RTGreen 的另一个优点是它的安全性。常规的 DNA 结合染料存在潜在的致突变性。基于这方面考虑，我们研发设计了 RTGreen 染料，它不能透过细胞膜，因而不能与活细胞的基因组DNA结合，确保了其安全性。然而，其他所有的商业性PCR荧光染料都可以在几分钟内进入细胞，例如 SYBR Green I，是一种众所周知的环境毒性比溴化乙锭 (EB) 还要大的诱变剂。
产品特性：
1. 极高的灵敏度：在推荇浓度下使用时可以获得最强的 PCR 扩增信号。
2. PCR抑制性极小：“按要求释放” 染料与 DNA 结合，使得 RTGreen 对 PCR 的抑制远小于 SYBR Green l。
3. 与快速 PCR兼容：对 PCR干扰极小，从而极大的缩短了PCR延伸时间。
4. 非常适合HRM分析：无“染料重分布”缺陷，兼容PCR后的高分辦率熔解曲线 (HRM) 分析。
5. 兼容多重PCR：在推荐浓度下使用时，无扩增子之间的染料迁移现象。
6. 超强稳定性：在大部分生化条件下非常稳定，可在室温下储存并可反复冻融。
7. 安全性好：细胞膜穿透性测试表明，RTGreen几乎不能穿透细胞膜，安全性高。
8. 优越的兼容性：和 SYBR Green I 光谱相似，兼容众多的 qPCR仪。无需改变任何您目前使用的操作步骤和仪器设备。
推荐PCR体系：
建议 PCR反应体系 (50μL)：使用时根据引物和模板不同调节到最佳。以下体系是非热启动的Taq酶体系，推荐使用 1× 的RTGreen。
试剂
| 混合液中终浓度
| 每种 dNTP (2mM)，各加 5μL
| 每种 0.2mM
| ddH₂O，定容至 50μL
| 定容调节终体积
| 10×无镁聚合酶缓冲液，5μL
| 1× | 引物 (终浓度) 各 0.1–1μM
| 0.1-1μM | Taq DNA 聚合酶 1–5单位
| 每个反应 1–5单位
| | 2.5μL of 50mM MgCl₂ | 2.5 mM | | 2.5μL of 20× RTGreen | 1× |
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RTGreen is a dye for quantitative real-time PCR (qPCR). It is a novel DNA-binding dye specially designed for qPCR and high-resolution melting (HRM) analysis. Via an "on-demand release" mechanism, it greatly reduces inhibition of PCR amplification. RTGreen outperforms SYBR Green I in many aspects. While possessing similar spectral properties, RTGreen differs from SYBR Green I with three key characteristics:
1. It exhibits much lower PCR inhibition than SYBR Green I, enabling qPCR experiments with fast PCR protocols.
2. It can be applied at higher concentrations in assays to generate significantly stronger amplification signals than SYBR Green I. Higher concentrations of RTGreen eliminate the "dye redistribution" artifact, making RTGreen suitable for both multiplex PCR and high-resolution melting (HRM) analysis.
3. This assay is increasingly applied for post-PCR genotyping and heteroduplex analysis.
Another advantage of RTGreen is its safety. Conventional DNA-binding dyes carry potential mutagenicity. With this concern in mind, we developed RTGreen. It cannot penetrate cell membranes and therefore cannot bind to genomic DNA in live cells, guaranteeing its safety. In contrast, all other commercial fluorescent PCR dyes can enter cells within minutes. For instance, SYBR Green I is a well-known mutagen with greater environmental toxicity than ethidium bromide (EB).
Product Features:
1. Ultra-high sensitivity: Delivers maximum PCR amplification signal when used at the recommended concentration.
2. Minimal PCR inhibition: The "on-demand release" mode of dye-DNA binding results in far less PCR inhibition by RTGreen compared with SYBR Green I.
3. Compatible with fast PCR: Minimal interference with PCR, which greatly shortens PCR extension time.
4. Ideal for HRM analysis: Free from the "dye redistribution" artifact, compatible with post-PCR high-resolution melting (HRM) analysis.
5. Compatible with multiplex PCR: No dye migration among amplicons at the recommended working concentration.
6. Excellent stability: Highly stable under most biochemical conditions, storable at room temperature and tolerant to repeated freeze-thaw cycles.
7. High safety: Cell membrane penetration assays demonstrate that RTGreen barely crosses cell membranes.
8. Excellent compatibility: Similar spectrum to SYBR Green I, compatible with most qPCR instruments. No modifications to your existing protocols or equipment are required.
Recommended PCR System:
Suggested PCR reaction system (50 μL): Optimize according to different primers and templates. The system below is designed for non-hot-start Taq polymerase, with 1× RTGreen recommended.
| Reagent | Final concentration in the mixture | | 5μL each of 2mM dNTP | 0.2mM each | | ddH₂O to a final volume of 50μL | Adjust to final volume | | 5μL of 10× polymerase buffer without magnesium | 1× | | 0.1-1μM each of primers (final concentrations) | 0.1-1μM | | 1-5 units of Taq DNA polymerase | 1-5 units per reaction | | 2.5μL of 50mM MgCl₂ | 2.5 mM | | 2.5μL of 20× RTGreen | 1× |
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ZJ26F0939511
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