DAPI染DNA的原理及使用方法

DAPI  即4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole)，分子式為C16 H15 N5 ·2C3 H6 O3 ，分子量457.48。

DAPI 是一種能夠與DNA強力結合的熒光染料。它結合到雙鏈DNA小溝的AT堿基對處，一個DAPI分子可以占據三個堿基對的位置。結合到雙鏈DNA上DAPI分子的熒光強度提高大約20倍，常用與熒光顯微鏡觀測，根據熒光的強度可以確定DNA的量。另外，因為DAPI可以透過完整的細胞膜，它可以用于活細胞和固定細胞的染色。

在熒光顯微鏡觀察下，DAPI染劑是利用紫外光波長的光線激發。單獨DAPI的最大吸收波長為340nm，最大發射波長為488nm；當DAPI與雙鏈DNA結合時，最大吸收波長為364nm，最大發射波長為454nm（10 mM Tris pH7.0，100 mM NaCl，10 mM EDTA），其發散光的波長范圍含蓋了藍色至青綠色。DAPI也可以和RNA結合，但產生的熒光強度只有與DNA結合的熒光強度的1/5，其發散光的波長范圍約在500nm左右。

DAPI的配制及貯存

固體粉末 ：避光，2-8 ℃保存，3年沒有問題。

貯存液 ：用無菌水配制成濃度1 mg/ml 的貯存液，配好后用錫紙包起來，避光，可在-20 ℃下長期保存。（DAPI易溶于水，在PBS中溶解度不高）

使用濃度 ：貯存液用1xPBS稀釋到終濃度100 ng/ml。

熒光封片液 ：0.5 mol/L碳酸鹽緩沖液與甘油等體積混合，pH9.5

染色與觀察 ：制好的玻片上滴加幾滴DAPI染液，染色10分鐘，流水沖去染液，濾紙吸除多余水分，加一滴熒光封片液，置于熒光顯微鏡下觀察，激發波長360-400nm。

注意事項 ：DAPI可能具有致癌性，全部操作過程中必須帶塑料或乳膠手套。

推薦廠商 ：Invitrogen，Sigma

結構式 ：

以下英文介紹摘自Wikipedia 。

DAPI or 4',6-diamidino-2-phenylindole is a fluorescent stain that binds strongly to DNA. It is used extensively in fluorescence microscopy. Since DAPI will pass through an intact cell membrane, it may be used to stain both live and fixed cells.

For fluorescence microscopy, DAPI is excited with ultraviolet light. When bound to double-stranded DNA its absorption maximum is at 358 nm and its emission maximum is at 461 nm. (This emission is fairly broad, and appears blue/cyan.) DAPI will also bind to RNA, though it is not as strongly fluorescent. Its emission shifts to around 500 nm when bound to RNA.

DAPI's blue emission is convenient for microscopists who wish to use multiple fluorescent stains in a single sample. There is fluorescence overlap between DAPI and green-fluorescent molecules like fluorescein and green fluorescent protein (GFP), or red-fluorescent stains like Texas Red, but using spectral unmixing or taking images sequentially can get around this.

Apart from labelling cell nuclei, the most popular application of DAPI is in detection of mycoplasma or virus DNA in cell cultures.

Because DAPI easily enters live cells and binds tightly to DNA, it is toxic and mutagenic. Care should be taken in its handling and disposal.

The Hoechst stains are similar to DAPI in that they are also blue-fluorescent DNA stains which are compatible with both live- and fixed-cell applications