DNA Quantification with PreciseGreen®
PreciseGreen DNA quantification solution is an ultrasensitive reagent to measure concentration of double-stranded DNA if that cannot be determined by measuring the absorbance at 260 nm. PreciseGreen selectively binds to double-stranded DNA, so nucleotides, single-stranded DNA, RNA, proteins, and other impurities do not impede the measurements. The dye bound to double-stranded DNA absorbs light with maximum at 503 nm and emits light with maximum at 525 nm. To detect the assay read-out, any type of fluorometers or fluorescence plate readers can be used.
The range of linearity for DNA concentration measurements with PreciseGreen is 1 pg/μL — 5 ng/μL. For precise reproducible fluorescence measurements, we recommend to dilute DNA and PreciseGreen in TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). To calculate DNA concentration we recommend first to build a calibration curve using a series of DNA standard dilutions.
Lumiprobe sells PreciseGreen DNA quantification solution in various packs and PreciseGreen DNA quantification kit. The kit includes both buffer and DNA standard stock solution in addition to the PreciseGreen solution. The amount of dye solution supplied with the kit is sufficient to analyze 200 experimental data points in a standard fluorometric cuvette (volume 3.5 ml), if the assay volume is equal to 2 ml. The assay can be rescaled if other types of equipment are used to detect fluorescence. Recommended assay volumes for commonly used fluorometric equipment are provided in the table below.
Protocol
1. Preparation of PreciseGreen working solution
Thaw the content of PreciseGreen dye vial, mix thoroughly, and dilute a needed amount of the PreciseGreen dye solution 200-fold with a buffer (we recommend to use 10 mM Тris HCl, 1 mM EDTA, pH 7.5). Mix and use within 3 hours. The volume of PreciseGreen working solution for each experimental data point should be equal to 50% of assay volume (check the table below to find the recommended volumes for your type of fluorometric equipment). Prepare enough PreciseGreen working solution for all experimental data points (for all samples and all DNA standard dilutions that you plan to analyze). Include also additional 10-25% of PreciseGreen working solution volume to exclude possible pipetting errors. To calculate the volume of diluted PreciseGreen, you can use the following formula:
VPreciseGreen = 5/8 × Vassay × (Nsamples + Nstandards), where Vassay is the assay volume of a sample or a standard, mL, Nsamples is the number of samples that you plan to analyze, and Nstandards is the number of standards that you plan to analyze (including blank sample).
2. Preparation of sample solution
Dilute your DNA sample in the buffer to get the volume of solution equal to 50% of assay volume (you may take any amount of DNA). Add equal volume of PreciseGreen working solution. Mix and incubate for 5 min. Likewise, prepare dilutions of DNA standard. Please, note that the dilutions of DNA standard should be within the range of DNA concentrations in your sample. The DNA standard stock solution is only provided with PreciseGreen DNA quantification kit. The users of PreciseGreen DNA quantification solution should use their own DNA standards.
Recommended volumes for DNA quantification with PreciseGreen DNA quantification solution:
| Type of equipment | Assay volume | Volume of diluted PreciseGreen | Volume of diluted DNA |
|---|---|---|---|
| Standard fluorometric cuvette (3.5 ml ) | 2 ml | 1 ml | 1 ml |
| Other fluorometric cuvettes | About 75% of cuvette volume | 37.5% of cuvette volume | 37.5% of cuvette volume |
| 96-well plate*, per well | 0.2 ml | 0.1 ml | 0.1 ml |
| 24-well plate, per well | 1 ml | 0.5 ml | 0.5 ml |
| Other plates | About 75% of well volume | 37.5% of well volume | 37.5% of well volume |
| NanoDrop™ 3300* | 0.1 ml | 0.05 ml | 0.05 ml |
* To maintain accuracy and precision of your measurements, we recommend to avoid pipetting volumes below 2 µL.
3. Fluorescence measurements
Measure fluorescence from standard and sample DNA solutions using appropriate absorption and emission wavelengths or filters (double-stranded DNA bound PreciseGreen dye absorbs light with maximum at 503 nm and emits light with maximum at 525 nm).
4. Calculation of DNA concentration
Plot fluorescence vs concentration
and apply linear regression function in any software to obtain linear equation reflecting fluorescence
(FL) vs concentration (C) dependence:
FL = A × C + B.
To calculate
DNA concentration in the diluted sample use the following formula:
Csample =
(FLsample — B)/A, where FLsample is sample solution
fluorescence.
To calculate DNA concentration in your undiluted sample use the following formula:
Сinit = Vassay × Csample / Vinit, where
Vassay is the assay volume, mL and Vinit is the volume of your
initial DNA sample, µL
Alternatively, you can use our dsDNA quantification and dilution calculators to have all necessary calculations done.
Linear regression example: fluorescence versus DNA concentration
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