DNA quantification with Pico488
Pico488 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. Pico488 selectively binds to double-stranded DNA, so nucleotides, single-stranded DNA, RNA, proteins, and other impurities do not impede the measurements. The structure of the dye is identical to that of PicoGreen™ dye. Double-stranded DNA bound Pico488 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 Pico488 is 1 pg/μL – 5 ng/μL. For precise reproducible fluorescence measurements, we recommend to dilute DNA and Pico488 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 Pico488 DNA quantification solution in various packs and Pico488 DNA quantification kit. The kit includes both buffer and DNA standard stock solution in addition to the Pico488 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.
1. Preparation of Pico488 working solution
Thaw the content of Pico488 dye vial, mix thoroughly, and dilute a needed amount of the Pico488 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 Pico488 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 Pico488 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 Pico488 working solution volume to exclude possible pipetting errors. To calculate the volume of diluted Pico448, you can use the following formula:
VPico488 = 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 Pico488 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 Pico488 DNA quantification kit. The users of Pico488 DNA quantification solution should use their own DNA standards.
Recommended volumes for DNA quantification with Pico488 DNA quantification solution:
|Type of equipment||Assay volume||Volume of diluted Pico488||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 Pico488 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