Technical Note 157

dsDNA Broad Range Assay Performance Data

Introduction

DeNovix® Fluorescence Assays enable dsDNA quantitation over a broad range of concentrations through the use of three distinct fluorescence assay kits.

The DeNovix dsDNA Broad Range Assay is ideal for for measuring sample concentrations of 0.1 – 2000 ng/µL, with an upper extended range of 4000 ng/µL. The assay enables quantitation of dsDNA concentrations in the standard detection range when using 1 – 20 µL sample volumes in a 200 µL total assay volume. The upper detection limit can be extended by adding 1 µL of a 4000 ng/µL sample to 199 µL of reagent.

The assay dye has an excitation/emission maxima of 350/460 in the presence of dsDNA and is selective for dsDNA over RNA, ssDNA and protein. The assay is compatible with fluorescence microplate readers and fluorometers with the appropriate excitation sources and emission detectors. The excitation and emission spectra are shown in Figure 1.

This technical note presents typical performance data for the DeNovix dsDNA Broad Range Assay measured on a DeNovix DS-11 FX Fluorometer.

Figure 1: Excitation and emission spectra for the DeNovix dsDNA Broad Range Quantitation Reagent in the Presence of Excess dsDNA.

Materials and Methods

A series of dilutions of calf thymus dsDNA was prepared in TE buffer. The assay working solution was prepared by mixing 10 mL of the assay buffer with  100 µL of the dye and 100 µL of the assay enhancer solution.

For each sample, 190 µL of the working solution was added to a thin-walled, clear UV-transparent 0.5 mL PCR tube (DeNovix cat #TUBE-PCR-0.5-500). 10 μL of dsDNA was added to each tube for samples with concentrations between 0.5 and 200 ng/μL. For the lower concentration samples, volumes were adjusted to 180 μl working solution and 20 μL of dsDNA. For concentrations above 200 ng/μL, volumes used were 199 μL of working solution and 1 μL of sample.

Reaction solutions were mixed and incubated at room temperature for 5 minutes. Three replicates of each sample were then measured on three separate DeNovix DS-11 FX Fluorometers.

Linearity

The linear response of measured dsDNA as a function of expected concentration in the standard linear range is presented in Figure 2.

Figure 2: Average measured dsDNA across the standard range of the DeNovix dsDNA Broad Range Assay.

Performance Results

Concentration of dsDNA measured through the core linear and extended concentration ranges for the DeNovix Broad Range Assay are shown below in Table 1.

The data presented in Table 1 and graphically represented in Figure 2 demonstrate that the DeNovix dsDNA Broad Range Fluorescence Assay enables measurement of dsDNA through a range of 0.1 – 4000 ng/μL by varying mass and volume of samples.

Table 1: DeNovix dsDNA Broad Range Assay Results Measured on a DeNovix DS-11 FX
Expected dsDNAMeasured dsDNA 
ng/μLng/μLStDev
0.10.1160.005
0.20.2370.011
0.50.5420.0
11.120.015
22.390.011
6.256.650.020
12.513.480.017
2526.830.030
5053.750.051
100106.10.094
200195.70.274
400358.080.263
10001050.920.071
20001974.530.134
30003123.240.248
40003551.610.353

Competitive Advantage

The DeNovix Broad Range Assay has a wider dynamic range than the comparable Qubit® Broad Range Assay. Figure 3 represents the high concentration range advantage of the DeNovix Broad Range Assay as compared to the Qubit® Assay.

Figure 3: Comparison of the DeNovix dsDNA Broad Range Assay and the Qubit® dsDNA Broad Range Assay.

Summary

The DeNovix dsDNA Broad Range Assay enables  specific, highly sensitive dsDNA quantification across a wide dynamic range.

If the Broad Range Assay does not cover the concentration range of your samples, consider using one of the alternate DeNovix dsDNA Assay Kits listed in Table 2.

DeNovix Fluorescence AssayRange
dsDNA Broad Range0.1 – 2000 ng/µL
dsDNA High Sensitvity5 pg/µL – 250 ng/µL
dsDNA Ultra High Sensitivity0.5 – 300 pg/µL
RNA Assay0.25 - 1500 ng/µL

Qubit® is a registered trademark of Thermo Fisher Scientific and its subsidiaries.

Revised 19 Oct 2020