DAPI staining

Summary

Cell staining with DAPI

Principle

DAPI is 4',6diamidino-2-phenylindole (4',6-diamidino-2-phenylindole) binds to the minor groove of double-stranded DNA, especially AT bases, and can also be inserted into fewer than three consecutive AT base pairs in a DNA sequence. When it binds to double-stranded DNA, the fluorescence intensity is enhanced 20-fold, whereas there is no fluorescence enhancement when it binds to single-stranded DNA.

Appliance

cell staining

Operation method

Principle and use of DAPI for DNA staining

Materials and Instruments

DAPI is 4',6-diamidino-2-phenylindole (4',6-diamidino-2-phenylindole), molecular formula C16 H15 N5 -2C3 H6 O3, molecular weight 457.48.
DAPI is a fluorescent dye that binds strongly to DNA. It binds to the AT base pair in the minor groove of double-stranded DNA, and one DAPI molecule can occupy three base pairs. The fluorescence intensity of a DAPI molecule bound to double-stranded DNA is increased about 20-fold, and is often observed with a fluorescence microscope, where the amount of DNA can be determined based on the intensity of the fluorescence. In addition, because DAPI can pass through intact cell membranes, it can be used for staining both living and fixed cells.
Under fluorescence microscope observation, DAPI stains are excited using light at ultraviolet wavelengths. DAPI alone has a maximum absorption wavelength of 340 nm and a maximum emission wavelength of 488 nm; when DAPI is combined with double-stranded DNA, it has a maximum absorption wavelength of 364 nm and a maximum emission wavelength of 454 nm (10 mM Tris pH 7.0, 100 mM NaCl, 10 mM EDTA), and the wavelength range of the evanescent light encompasses the blue to the lime green. DAPI can also bind to RNA, but the fluorescence intensity produced is only 1/5 of the fluorescence intensity of the fluorescence bound to DNA, and the wavelength range of its emitted light is about 500 nm.

Move

Preparation and storage of DAPI
Solid powder ：Protected from light, stored at 2-8 ℃, no problem for 3 years.
Storage solution: Prepare the storage solution with sterile water at a concentration of 1 mg/ml, wrap it with tin paper after preparation, protect it from light, and store it at -20 ℃ for a long time. (DAPI is easily soluble in water, and its solubility in PBS is not high.) Concentration: Dilute the storage solution with 1xPBS to a final concentration of 100 ng/ml. fluorescent sealing solution: 0.5 mol/L carbonate buffer mixed with glycerol in equal volume, pH 9.5 Staining and observation: Add a few drops of DAPI staining solution on the prepared slides and stain them for 10 minutes, rinse off the staining solution by running water and remove the excess water with filter paper, then add one drop of fluorescent sealing solution to the slides. Add a drop of fluorescent sealing solution and observe under a fluorescence microscope with an excitation wavelength of 360-400 nm. Precautions: DAPI may be carcinogenic, so plastic or latex gloves must be worn during all operations. Recommended Manufacturer: Invitrogen, Sigma Structural formula: The following description is taken from 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. 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. 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 has been in a single sample. 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. 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.

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