Many instruments for quantification are now making calculations automatically and the quantitation calculator can be already embedded into the reader. In case these calculations are not automated in your instrument the Lumiprobe dsDNA quantification calculator can be used.
Below are some easy steps to perform quantification in plates with Pico488 quantification kit by Lumiprobe (analog of Quant-iT® PicoGreen® dsDNA Assay Kit). In case dsDNA standards are available only the quantification reagent can be used.
1. Prepare the 1x TE buffer by diluting the 20x TE concentrate 1:20 by deionized water.
2. Prepare the Dye working solution by diluting the Pico488 reagent 1:200 with 1x TE buffer (you need about 200μL of Pico488 working solution for each standard and sample).
3. Make the DNA standards for quantification. The following scheme of dilution can be used as an example:
Standard 1: Prepare a E. coli DNA standard solution with concentration 5 ng/µL: 30 µL of the DNA standard (100 ng/µL) should be mixed with 570 µL of 1× TE buffer.
Standard 2: Prepare a E. coli DNA standard solution with concentration 0.5 ng/µL: 3 µL of the DNA standard (100 ng/µL) should be mixed with 597 µL of 1× TE buffer.
Standard 3: Prepare a E. coli DNA standard solution with concentration 0.05 ng/µL: 60 µL of the DNA Standard 2 (0.5 ng/µL) should be mixed with 540 µL of 1× TE buffer.
In case other concentarations are preffered dilution calculator can be used.
4. Set up microplates for fluorescence-based assays.
5. Prepare standards and samples for concentration measurement. For a more accurate quantitative analysis, measurements can be made twice.
To prepare standards for concentration measurements, mix in wells of microplate 180 μl of the Pico488 working solution and:
20 μl of 1x TE buffer (0 ng / mkl),
20 μl of each DNA standard prepared on step 3 (final concentration in these experiments will be 0.5-0.005 ng/μL)
6. To prepare samples for concentration measurements, mix in wells of microplate 190-199 μl of the Pico488 working solution and 20-1 mkl of DNA sample.
7. Incubate the plate on the shaker for 5 minutes at room temperature.
The next steps should be carried out according to the instructions of the microplate reader. After measurements it is necessary to determine the linear range of fluorescence dependence on concentration and build a calibration curve. The quantification calibration curve can be made manually or by Lumiprobe calculator. When measuring concentrations above 1 ng / μL (final concentration), you should ensure that the fluorescence level does not exceed the measuring range of the instrument. In case the dependence is not linear due to the high concentration the more diluted serie can be prepared to better fit the range of instrument.