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產(chǎn)品信息

產(chǎn)品簡(jiǎn)介：

EvaGreen 是一種用于實(shí)時(shí)定量 PCR（qPCR）的DNA結(jié)合染料。它的諸多優(yōu)點(diǎn)使它遠(yuǎn)勝于SYBR Green I。除了有相似的光譜特性，EvaGreen有三個(gè)主要特點(diǎn)使它區(qū)別于SYBR Green I。

EvaGreen對(duì)PCR的抑制性遠(yuǎn)小于SYBR Green I。因此，使用EvaGreen進(jìn)行的qPCR實(shí)驗(yàn)可以使用快速PCR步驟。同時(shí)，EvaGreen在實(shí)驗(yàn)中可以使用較高的濃度，從而獲得遠(yuǎn)強(qiáng)于SYBR Green I擴(kuò)增信號(hào)。較高濃度的EvaGreen也消除了“染料重分布”的缺陷，使EvaGreen既可用于多重PCR，也可用于高分辨率（高清晰）熔解曲線分析（HRM）。該分析正被越來(lái)越多的用于PCR后的基因分型和異源雙鏈分析。由于SYBR Green I對(duì)PCR的抑制性，從而要求其使用濃度必須很低，因此 SYBR Green I無(wú)法解決由低濃度造成的染料重分布問(wèn)題，既不能用于多重PCR也不能用于HRM。同時(shí)，染料重分布問(wèn)題也可能影響常規(guī)熔解曲線的可靠性，因?yàn)榈腿埸c(diǎn)的DNA鏈可能由于這種原因而無(wú)法檢測(cè)到 。

EvaGreen的穩(wěn)定性極強(qiáng)。在正常的儲(chǔ)存、操作和PCR過(guò)程中不會(huì)被破壞。在緩沖溶液中的染料可以安全的儲(chǔ)存在室溫或冰箱里，也可以反復(fù)凍融。與之相反，SYBR Green I不穩(wěn)定而且降解后對(duì)PCR抑制性更強(qiáng) 。

EvaGreen降低了細(xì)胞膜透性，因而比SYBR Green I更加安全。測(cè)試結(jié)果顯示，EvaGreen既沒(méi)有誘變性也沒(méi)有細(xì)胞毒性。相反，雖然 SYBR Green I本身誘變性很弱，但它在細(xì)胞中可能抑制了正常DNA的修復(fù)機(jī)制，使其有誘變?cè)鰪?qiáng)作用。

EvaGreen的特性：

1. 極高的靈敏度：在推薦濃度下使用時(shí)可以獲得最強(qiáng)的PCR擴(kuò)增信號(hào)。

2. PCR 抑制性極?。褐悄芑?span>“按要求釋放”DNA結(jié)合技術(shù)使得EvaGreen對(duì)PCR的抑制遠(yuǎn)小于SYBR Green I。

3. 和快速PCR兼容：對(duì)PCR干擾極小，從而極大的縮短了PCR延伸時(shí)間。

4. 非常適合HRM分析：無(wú)“染料重分布”缺陷，兼容PCR后的高分辨率熔解曲線（HRM）分析。

5. 兼容多重 PCR：在推薦濃度下使用時(shí)，無(wú)擴(kuò)增子之間的染料遷移現(xiàn)象。

6. 超強(qiáng)穩(wěn)定性：在大部分生化條件下非常穩(wěn)定，可在室溫下儲(chǔ)存并可反復(fù)凍融。

7. 安全性好：細(xì)胞膜穿透性測(cè)試表明，EvaGreen幾乎不能穿透細(xì)胞膜，安全性高。

8. 優(yōu)越的兼容性：和SYBR Green I光譜相似，和各知名品牌的qPCR儀器兼容。用EvaGreen 替代SYBR Green I，無(wú)需改變?nèi)魏文壳笆褂玫牟僮鞑襟E和儀器設(shè)備。

英文版

EvaGreen 20× in water ； Concentration: 20× (25 uM) in water

Color and Form: Light orange solution

Product Description

EvaGreen is a very sensitive dye for the detection of double stranded DNA (dsDNA). The dye is a green fluorescent nucleic acid dye with features that make it useful for non-specific detection of amplification in realtime qPCR experiments.

Compared with the widely used SYBR Green I, EvaGreen dye is generally less inhibitory toward PCR and less likely to cause nonspecific amplification , EvaGreen dye can be used at a much higher dye concentration than SYBR Green I, resulting in more robust PCR signal.

The PCR reaction can be monitored using our existing optical setting for SYBR Green I or FAM on any commercial real-time PCR cycler. The qPCR protocol provided below is for PCR using regular non-hot-start Taq. Use of a hot-start Taq may require some adjustment of PCR buffer composition in terms of ionic strength and pH to best take the advantage of EvaGreen dye. The water soluble solvent such as DMSO or glycerol are frequently added to stabilize master mixes. These components and pH may need to be optimized depending on the enzyme used.

Protocol

Calculate the volumes of reagents required for the reaction.

1.On ice, prepare a 1x master mix containing no DNA, by mixing the components in the following order: water, Taq polymerase buffer, dNTPs, MgCl2, EvaGreen, Taq polymerase, and primers.

2.Transfer master mix to tubes or plates. Add DNA (50 ng per reaction).

3.Proceed with amplification according to your instrument manufacturer. Perform real-time PCR on a thermocycling fluorometer and record the fluorescence signal at the annealing or extension step.

Notes:

1) Always use positive and negative controls when doing qPCR experiments.

2) The temperature program for the qPCR amplification does not differ from standard PCR program for the given template and primers.

3) For the detection, FAM or FAM/SYBR channel should be used.

4) When using ABI Sequence Detection Systems, make sure to select NONE for the passive reference under the tab WELL INSPECTOR.

5) BSA may be required if the reaction is run on a Roche LightCycler. A final BSA concentration of 0.5 mg/mL may be sufficient. With SYBR Green, addition of a protein such as BSA results in a fluorescence increase, which provides a background signal that triggers the start of a LightCycler. Because EvaGreen dye is less sensitive to proteins, you may need to adjust the instrument setting (for background fluorescence) so that the instrument will start.