SYTO 9 (20× in DMSO) PCR用核酸染料

産品信息

産品描述

SYTO 9綠色熒光(guāng)核酸染料（SYTO 9 Green Fluorescent Nucleic Acid Stain），是一(yī)款優秀的細胞核和染色體複染劑，具細胞膜滲透性。SYTO 9高(gāo)親和結合DNA（以及RNA），一(yī)旦結合後呈現明顯增強的熒光(guāng)信号，用藍光(guāng)激發（最大發射波長(cháng)485nm），發綠色熒光(guāng)（最大激發分别是498nm（DNA）和501nm（RNA））。SYTO 9由于能(néng)很好的滲透進入原核和真核細胞膜，普遍用作核複染劑（特别是細菌細胞），常常與死細胞核複染劑（比如：碘化丙啶PI）聯合使用，用于活細菌/死細菌染色[1-2]。

更值得關注的是，SYTO 9作為(wèi)一(yī)款靈敏的DNA結合染料，在RT-PCR中，表現出許多(duō)優異的特征，包括：在寬廣的染料濃度下(xià)産生(shēng)高(gāo)度可重複的DNA熔解曲線，極低(dī)的PCR抑制率，高(gāo)信噪比和高(gāo)靈敏度，使其成為(wèi)一(yī)款理想的SYBR Green I替代染料，用于RT-PCR、DNA熔解曲線分析、以及環介導等溫擴增（LAMP）實驗[3-5]。

本品為(wèi)溶于DMSO的20×SYTO 9核酸染料，達PCR級别，使用時僅需根據體系加量使其終濃度為(wèi)1×即可。

保存與運輸方法

保存：2-8℃避光(guāng)保存，也可置于-20℃保存，2年(nián)有效。

運輸：冰袋運輸。

注意事(shì)項

1） 本品保存和使用過程中請注意避光(guāng)。

2） 本品使用前，請置于室溫回溫，之後用漩渦混勻器(qì)完全混勻。本品高(gāo)度穩定，但染料在保存的過程中會(huì)吸附到(dào)管壁上(shàng)，漩渦混勻數秒(miǎo)使其充分溶解。

3） 為(wèi)了您的安全和健康，請穿實驗服并戴一(yī)次性手套操作。

使用方法（RT-PCR）

1． 按照(zhào)以下(xià)表格配制反應體系（僅作參考，根據實際情況或參考文獻資料進行優化調整），

2. 根據檢測樣本數于冰上(shàng)配制足量不含DNA的2×反應體系（master mix），按照(zhào)以下(xià)順序混合各組分：水(shuǐ)→Taq polymerase buffer→dNTPs→MgCl2→SYTO 9→Hot-start Taq DNApolymerase。

3. 将以上(shàng)體系混勻後分裝至qPCR管或平闆内。根據下(xià)表來配制1×反應體系（master mix）。

4. 在合适的儀器(qì)内啓動qPCR反應并記錄退火或延伸步驟的熒光(guāng)信号。【可參考以下(xià)程序】

注意事(shì)項

4） 本品保存和使用過程中請注意避光(guāng)。

5） 本品使用前，請置于室溫回溫，之後用漩渦混勻器(qì)完全混勻。本品高(gāo)度穩定，但染料在保存的過程中會(huì)吸附到(dào)管壁上(shàng)，漩渦混勻數秒(miǎo)使其充分溶解。

6） 目前沒數據表明SYTO 9具緻畸性或毒性。由于該探針與核酸結合，可能(néng)被當作一(yī)種潛在的突變劑，需做适當的防護措施。由于DMSO能(néng)促進有機(jī)分子進入組織，此儲存液在使用的過程中務必妥善操作。根據當地的政策來處理使用本品後的廢液。

7） 為(wèi)了您的安全和健康，請穿實驗服并戴一(yī)次性手套操作。

應用示例（來自(zì)文獻）

一(yī)、LAMP實驗（文獻：PMID: 31681184; PMCID: PMC6803449）

1.1文章目的：比較23種DNA染料在實時LAMP檢測Salmonella Enteritidis (S. Enteritidis) strain的表現差異。

1.2 LAMP實驗方案：

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各染料在擴增抑制性上(shàng)的性能(néng)比較：

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實驗（文獻：PMID: 27886052; PMCID: PMC5133880）

2.1文章目的：基于熔解曲線的多(duō)重RT-qPCR實驗來同時檢測4種HCoVs。此文用SYTO 9替代SYBR Green I作為(wèi)核酸染料。

2.2 RT-PCR實驗方案：

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檢測靈敏度：

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

參考文獻

[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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