Microplate-based DNA Quantification with EzFluoroStain DNA reagent

Abstract

This protocol offers an safer alternative to the ethidium bromide-based DNA quantification protocol, utilizing an DNA-selective dye EzFluoroStain DNA (WSE-7130, ATTO corporation, Tokyo, Japan). This protocol allows to quantify about 2-1000 ng DNA/well, using a standard fluorescent plate reader.

Before start

We recommend to aliquot EzFluoroStain DNA reagent in ~10µL and minimize freeze-thaw cycles (several freeze-thaw cycles are OK). Before starting the experiment, thaw an aliquot of EzFluoroStain DNA reagent while protecting from light.

Steps

Sample preparation

Prepare dilution series of standard DNA solution, depending on the concentration of your sample.

Note

To make full-range standard curve (~4 ng/well to 1000 ng/well), prepare 200, 100, 50, 25, ... , 1.56, 0.78 ng/µL in Tris-EDTA buffer. For routine use, 3-4 points may be sufficient (e.g., 200, 20, 2ng/µL).

Prepare EzFluoroStain DNA working solution.

Note

Thaw an aliquot of EzFluoroStain DNA at room temperature and dilute EzFluoroStain DNA solution by 1000-fold in Tris-EDTA buffer and mix by vortex. Prepare required amount of the solution (at least 95 µL/well is needed).

Mix 5 µL of the DNA samples with 95 µL of the EzFluoroStain DNA working solution on a black 96-well microplate.

Note

The volume of the DNA sample can be varied depending on the estimated concentration of the sample. 1 µL/well (for higher concentration samples) to 10 µL/well (for lower concentration samples) may be used. Please adjust the volume of the working solution so that the total volume becomes 100 µL (for standard 96-well plates). Although the fluorescence can be measured soon after mixing the solution, it might be better to wait for 3-5 min to equilibrate the dye-DNA binding. The fluorescence is stable at least for 30 min at room temperature. Although all-black microplates are recommended for higher sensitivity, standard clear microplates may also be used.

Measurement & analysis

Measure fluorescence.

Note

The excitation/emission maximum are reported to be around 495 nm and 522 nm. For filter-based fluorimeter, excitation filter around 475 nm and band-pass emission filter 500-550 nm is recommended. For monochrometer-based fluorimeter, it would be better to use the maximum wavelength for excitation/emission settings.

Calculate the concentration of your samples using standard curve.

Abstract

Before start

Steps

Sample preparation

Measurement & analysis

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