产品简介：
SYBR Green I is a very sensitive dye for the detection of double stranded DNA (dsDNA), So it has been widely used in non-specific detection of amplification in realtime qPCR experiments. The double-strand DNA-specific SYBR Green I fluorescent reporter offers distinct advantages. SYBR Green I dye is inexpensive, easy to use, and sensitive. Well-designed primers must be used in SYBR Green quantitative RT-PCR reactions because SYBR Green I dye will detect nonspecific products, resulting in an overestimation of the target concentration.
操作步骤：
1. The flowing table 1 is our SYBR Green I RT-PCR Reagents Kit recipe for reference only. Please optimize it by yourself.
2. On ice, prepare a 2x master mix containing no DNA, by mixing the components in the following order: water, DMSO, Taq polymerase buffer, dNTPs, MgCl2, Sybr Green, Taq polymerase.
3. Transfer 2x QuantiTect SYBR Green PCR Master Mix to tubes or plates. Add RNase-free water. Mix the individual solutions.
4. Prepare a reaction mix according to Table 2.
Due to the hot start, it is not necessary to keep samples on ice during reaction setup or while programming the real-time cycler.
Program your real-time cycler according to the program outlined in Table 3
本产品为10000倍预混液。SYBR Green I 与dsDNA 结合荧光信号可增强800～1000倍。在PCR反应体系中，加入过量SYBR Green I 荧光染料，它特异性地掺入DNA双链后，荧光信号增强，而不掺入链中的SYBR Green I 染料分子荧光不变，从而保证荧光信号的增加与PCR产物的增加完全同步。荧光可以在退火阶段或者延伸阶段测定。
使用浓度对荧光PCR结果的影响
SYBR Green I 的使用浓度是保证荧光定量PCR实验成功非常关键的因素。如果SYBR Green I 的浓度过低会使荧光信号的变化降低，这就意味着低拷贝的样品可能无法检出。而在高浓度时，将会抑制PCR反应，降低PCR反应效率。所以一般在使用SYBR Green I 时应根据实际情况优化使用浓度，反应的终浓度为1×到0.2×之间。
Mg2＋浓度的影响
提高Mg2＋浓度可以降低SYBR Green I对PCR反应的抑制作用。我们建议在用SUPER Green I进行荧光PCR反应时，Mg2＋浓度比无SUPER Green I 的普通 PCR 反应高出0.5～3mM。
| Reagent | Final concentration in the mix |
| dNTP | 0.25mM |
| Tween20 | 1% |
| BSA | 0.1% vol. |
| Tris(pH8.4) | 50mM |
| Hot-start Taq DNA polymerase | 1.25 u per reaction |
| NH4Cl | 10mM |
| KCl | 20mM |
| MaCl2 | 2.5mM |
| Sybr Green | 2X |
2. On ice, prepare a 2x master mix containing no DNA, by mixing the components in the following order: water, DMSO, Taq polymerase buffer, dNTPs, MgCl2, Sybr Green, Taq polymerase.
3. Transfer 2x QuantiTect SYBR Green PCR Master Mix to tubes or plates. Add RNase-free water. Mix the individual solutions.
4. Prepare a reaction mix according to Table 2.
Due to the hot start, it is not necessary to keep samples on ice during reaction setup or while programming the real-time cycler.
| PCR Reaction Setup: Table 2 | |
| DNA | Template DNA (<500 ng/reaction) |
| SYBR Mix | 25.0 μL |
| Primer1（引物1） | 2ul（5uM) |
| Primer2（引物2） | 2ul（5uM） |
| dd H2O | Added to 50.0 μL |
Program your real-time cycler according to the program outlined in Table 3
| PCR initial activation step |
Heating cycling Number of cycles 40 |
| 95 °C 5min | 96 °C 10s 60 °C 30s |
本产品为10000倍预混液。SYBR Green I 与dsDNA 结合荧光信号可增强800～1000倍。在PCR反应体系中，加入过量SYBR Green I 荧光染料，它特异性地掺入DNA双链后，荧光信号增强，而不掺入链中的SYBR Green I 染料分子荧光不变，从而保证荧光信号的增加与PCR产物的增加完全同步。荧光可以在退火阶段或者延伸阶段测定。
使用浓度对荧光PCR结果的影响
SYBR Green I 的使用浓度是保证荧光定量PCR实验成功非常关键的因素。如果SYBR Green I 的浓度过低会使荧光信号的变化降低，这就意味着低拷贝的样品可能无法检出。而在高浓度时，将会抑制PCR反应，降低PCR反应效率。所以一般在使用SYBR Green I 时应根据实际情况优化使用浓度，反应的终浓度为1×到0.2×之间。
Mg2＋浓度的影响
提高Mg2＋浓度可以降低SYBR Green I对PCR反应的抑制作用。我们建议在用SUPER Green I进行荧光PCR反应时，Mg2＋浓度比无SUPER Green I 的普通 PCR 反应高出0.5～3mM。
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参考文献 & 客户发表文献
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摩尔浓度计算公式
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