SYTO 9 (20× in DMSO) PCR用核酸染料

产品信息

产品描述

SYTO 9绿色荧光核酸染料（SYTO 9 Green Fluorescent Nucleic Acid Stain），是一款优秀的细胞核和染色体复染剂，具细胞膜渗透性。SYTO 9高亲和结合DNA（以及RNA），一旦结合后呈现明显增强的荧光信号，用蓝光激发（最大发射波长485nm），发绿色荧光（最大激发分别是498nm（DNA）和501nm（RNA））。SYTO 9由于能很好的渗透进入原核和真核细胞膜，普遍用作核复染剂（特别是细菌细胞），常常与死细胞核复染剂（比如：碘化丙啶PI）联合使用，用于活细菌/死细菌染色[1-2]。

更值得关注的是，SYTO 9作为一款灵敏的DNA结合染料，在RT-PCR中，表现出许多优异的特征，包括：在宽广的染料浓度下产生高度可重复的DNA熔解曲线，极低的PCR抑制率，高信噪比和高灵敏度，使其成为一款理想的SYBR Green I替代染料，用于RT-PCR、DNA熔解曲线分析、以及环介导等温扩增（LAMP）实验[3-5]。

本品为溶于DMSO的20×SYTO 9核酸染料，达PCR级别，使用时仅需根据体系加量使其终浓度为1×即可。

保存与运输方法

保存：2-8℃避光保存，也可置于-20℃保存，2年有效。

运输：冰袋运输。

注意事项

1） 本品保存和使用过程中请注意避光。

2） 本品使用前，请置于室温回温，之后用漩涡混匀器完全混匀。本品高度稳定，但染料在保存的过程中会吸附到管壁上，漩涡混匀数秒使其充分溶解。

3） 为了您的安全和健康，请穿实验服并戴一次性手套操作。

使用方法（RT-PCR）

1． 按照以下表格配制反应体系（仅作参考，根据实际情况或参考文献资料进行优化调整），

2. 根据检测样本数于冰上配制足量不含DNA的2×反应体系（master mix），按照以下顺序混合各组分：水→Taq polymerase buffer→dNTPs→MgCl2→SYTO 9→Hot-start Taq DNApolymerase。

3. 将以上体系混匀后分装至qPCR管或平板内。根据下表来配制1×反应体系（master mix）。

4. 在合适的仪器内启动qPCR反应并记录退火或延伸步骤的荧光信号。【可参考以下程序】

注意事项

4） 本品保存和使用过程中请注意避光。

5） 本品使用前，请置于室温回温，之后用漩涡混匀器完全混匀。本品高度稳定，但染料在保存的过程中会吸附到管壁上，漩涡混匀数秒使其充分溶解。

6） 目前没数据表明SYTO 9具致畸性或毒性。由于该探针与核酸结合，可能被当作一种潜在的突变剂，需做适当的防护措施。由于DMSO能促进有机分子进入组织，此储存液在使用的过程中务必妥善操作。根据当地的政策来处理使用本品后的废液。

7） 为了您的安全和健康，请穿实验服并戴一次性手套操作。

应用示例（来自文献）

一、LAMP实验（文献：PMID: 31681184; PMCID: PMC6803449）

1.1文章目的：比较23种DNA染料在实时LAMP检测Salmonella Enteritidis (S. Enteritidis) strain的表现差异。

1.2 LAMP实验方案：

The LAMP assay was carried out in 10 μl master mixture containing 0.2 μM of F3; 0.2 μM of B3; 1.4 μM of FIP; 1.4 μM of BIP; 0.8 μM of LF; 0.8 μM of LB; 1.4 mM dNTP mix (DNA Technology, Aarhus, Denmark), 0.5 M Betaine (Sigma-Aldrich, Denmark), 4 U of Bst 2.0 DNA polymerase (New England BioLabs), 1× isothermal amplification buffer (comprising 20 mM Tris–HCl, 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, and 0.1% Tween® 20, pH 8.8), various concentrations ranging from 0.5 μM to 10 μM of each dye that included SYTO 9, SYTO 13, SYTO 16, SYTO 24, SYTO 60, SYTO 62, SYTO 64, SYTO 82, SYBR Green I, SYBR Gold, YOPRO1, TOTO1, TOTO3, BOBO3, POPO3, and TOPRO3; Eva Green; Boxto; Miami Green, Miami Yellow, and Miami Orange, Pico 488and Nuclear Green DCS1, sterilized water and DNA template.

 The reactions were performed at 65°C for 60 min and the reactions were then terminated by heating to 90°C for 10 min. The fluorescent signal was recorded every minute of amplification. 

1.3各染料在扩增抑制性上的性能比较：

Fig. Comparison of Tt value against dye concentration for 20 dyes which exhibited fluorescence in real-time LAMP in presence of 2 ng DNA S. Enteritidis per reaction. The slope of the line indicates the degree of inhibition in real-time LAMP reaction.

According to the results of the real-time LAMP reaction and slopes of linear relationship of all these dyes, the inhibitory effect of these dyes on the real-time LAMP reaction was classified into four different groups: (1) non-inhibition effect, (2) medium inhibition effect, (3) high inhibition effect, and (4) very high inhibition effect (Figure 1 and Table 1).

二、RT-PCR实验（文献：PMID: 27886052; PMCID: PMC5133880）

2.1文章目的：基于熔解曲线的多重RT-qPCR实验来同时检测4种HCoVs。此文用SYTO 9替代SYBR Green I作为核酸染料。

2.2 RT-PCR实验方案：

The RT-qPCR was performed using QIAGEN OneStep RT-PCR Kit (QIAGEN, Hilden, Germany) with SYTO 9 as the fluorescent dye. Thirty μL reactions including 3 μL template input were run on a Light Cycler 96 RT-qPCR System (Roche Diagnostics, Mannheim, Germany). Reaction conditions were: 30 min RT at 50 °C, 15 min at 94 °C for inactivation of reverse transcriptase (RT), followed by 40 cycles of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 1 min. Melting curve analysis was performed under the condition of 95 °C for 60 s, 40 °C for 60 s, 65 °C for 1 s, then followed by a slow increase from 65 °C to 95 °C with a speed of 0.07 °C per second.

 Reaction conditions were: 30 min RT at 50 °C, 15 min at 94 °C for inactivation of reverse transcriptase (RT), followed by 40 cycles of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 1 min. Melting curve analysis was performed under the condition of 95 °C for 60 s, 40 °C for 60 s, 65 °C for 1 s, then followed by a slow increase from 65 °C to 95 °C with a speed of 0.07 °C per second.

2.3检测灵敏度：

Fig. Sensitivity of the multiplex RT-qPCR assay using serially diluted RNA stocks. PDs, primer dimers; NTC, non-template control.

参考文献

[1] Stiefel P, Schmidt-Emrich S, Maniura-Weber K, Ren Q. Critical aspects of using bacterial cell viability assays with the fluorophores SYTO9 and propidium iodide. BMC Microbiol. 2015 Feb 18;15:36. doi: 10.1186/s12866-015-0376-x. PMID: 25881030; PMCID: PMC4337318.

[2] McGoverin C, Robertson J, Jonmohamadi Y, Swift S, Vanholsbeeck F. Species Dependence of SYTO 9 Staining of Bacteria. Front Microbiol. 2020 Sep 3;11:545419. doi: 10.3389/fmicb.2020.545419. PMID: 33013779; PMCID: PMC7494787.

[3] Quyen TL, Ngo TA, Bang DD, Madsen M, Wolff A. Classification of Multiple DNA Dyes Based on Inhibition Effects on Real-Time Loop-Mediated Isothermal Amplification (LAMP): Prospect for Point of Care Setting. Front Microbiol. 2019 Oct 15;10:2234. doi: 10.3389/fmicb.2019.02234. PMID: 31681184; PMCID: PMC6803449.

[4] Monis PT, Giglio S, Saint CP. Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis. Anal Biochem. 2005 May 1;340(1):24-34. doi: 10.1016/j.ab.2005.01.046. PMID: 15802126.

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