PicoGreen

PicoGreen is a fluorescent dye used for quantifying double-stranded DNA in solution. Developed by Molecular Probes (now part of Thermo Fisher Scientific), PicoGreen binds specifically to double-stranded DNA and exhibits bright fluorescence upon binding. In solution, it has minimal fluorescence with single-stranded DNA or RNA, enabling selective dsDNA detection.

Mechanism and detection: The dye binds to the minor groove of double-stranded DNA; when bound, its fluorescence increases markedly. The assay is read with a fluorometer or microplate reader using excitation around 480 nm and emission around 520 nm. Signal is proportional to dsDNA concentration over a broad linear range, allowing quantification from small amounts of DNA.

Applications and protocol: PicoGreen is used to quantify dsDNA in purified samples, plasmid DNA, PCR products, restriction digests, and DNA extracts where accurate measurements are needed. A typical protocol involves mixing a small volume of PicoGreen reagent with the DNA sample in a buffer (often TE), incubating briefly in the dark, and measuring fluorescence against a dsDNA standard curve. Standards are prepared from known dsDNA concentrations to generate a reference for sample quantitation.

Limitations and considerations: The reagent can be affected by contaminants such as organic solvents or high salt; compatibility with buffers and detergents should be verified. It is not suitable for measuring single-stranded DNA, and the presence of RNA can contribute to background fluorescence under some conditions. Reagents should be stored and protected from light according to supplier guidelines. PicoGreen is commonly supplied as a dsDNA quantitation kit and is widely used in molecular biology workflows.