Quant-iT™ PicoGreen® dsDNA Assay Kit

Protocol tips

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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

DNA quantitation
The DNA concentration was quantified with three methods: absorbance measurement at 260 nm, fluorescence measurement with PicoGreen and qPCR with Alu-based assay.

Absorbance measurements at 260 nm were done using NanoDrop ND-1000 (Thermo Fisher Scientific Inc., Waltham, USA) with 1 μl of sample. This is the only method which also provided information about the purity of isolated DNA (using A260/280 ratio).

For DNA quantification with PicoGreen (PG), Quant-iT™ PicoGreen® dsDNA Reagents and Kits (Invitrogen – Molecular Probes, Eugene, OR, USA) were used. An aqueous working solution of PG reagent was freshly prepared as a 1:200 dilution of the concentrated DMSO solution to 1x TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 7.5) prepared from 20x TE supplied by the manufacturer. 10 μl of DNA samples diluted in 90 μl of 1x TE buffer were mixed with 100 μl PG working solution to reach a final volume of 200 μl. The fluorometric measurements were performed using Tecan Safire 2 (Tecan, Grödig, Austria) at λex 480 nm and λem 520 nm. Fluorescence of specimens was compared with fluorometric measurements from a standard sample. Lambda DNA supplied by the manufacturer served as standard sample at a stock concentration of 100 ng/μl. It was used to construct the standard curve by dilution to final concentrations of 2.5 ng/μl, 1 ng/μl, 500 pg/μl, 250 pg/μl, 100 pg/μl, 50 pg/μl, 25 pg/μl, 10 pg/μl and 5 pg/μl. The lowest possible concentration (fluorescence) of a standard sample that could be used was 25 pg/μl; therefore, points of the calibration curve under this concentration were excluded from the analysis.

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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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