Quant-iT™ PicoGreen® dsDNA Assay Kit

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	To minimize photo bleaching effects, keep the time for fluorescence measurement constant for all samples. During incubation at room temperature, protected from light.

Protocol tips
To minimize photo bleaching effects, keep the time for fluorescence measurement constant for all samples. During incubation at room temperature, protected from light.

Publication protocol

Measurement of circulating DNA level
DNA was extracted from serum samples and then quantified using a PicoGreen DNA detection kit (Invitrogen) according to the manufacturer's instructions . In briefly, DNA was extracted from 200 µl of serum samples using a QIAamp Blood Kit (Qiagene) using the blood and body fluid protocol as recommended by the manufacturer. After the removal of most proteins by digestion with proteinase K, the sample was applied to the QIAamp 96 plate. DNA was adsorbed onto the silica membrane during a brief centrifugation step, while any remaining protein, salt and other contaminants were completely removed by three consecutive washes. Membrane-bound DNA was then eluted in double deionized HO or Tris–EDTA buffer. A final elution volume of 200 µl was used. Quantification of DNA was carried out using a PicoGreen DNA detection kit (Invitrogen). Calf thymus DNA (100 mg/ml; Sigma-Aldrich) was used as the standard. The concentration of DNA in the standard curve ranged from 0 to 100 ng/ml. Briefly, 20 ml of final DNA eluated was mixed with 1 ml of Tris–EDTA (10 mmol/l Tris–HCl, 1 mmol/l EDTA, pH 7.5) diluted with PicoGreen reagent. Fluorescence intensity was measured on an F-2000 spectrofluorometer (Molecular Devices) at excitation wavelength of 480 nm and an emission of 520 nm. Standard curve used to determine the levels of circulating DNA in the samples was established by the linear relationship between the known concentrations of calf thymus DNA (Sigma-Aldrich) and the corresponding fluorescence intensities.



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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Quant-iT™ PicoGreen® dsDNA Assay Kit below.

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