TN 235 Thermo Fisher Qubit™ 1X dsDNA Assays Performance Data

Introduction

DeNovix DS-11 Series and QFX Fluorometers enable the accurate and specific quantification of nucleic acids, proteins and other biomolecules through fluorescence measurement. This technical note presents typical performance data measured on the DeNovix QFX using the Thermo Fisher Qubit™ (Qubit™) 1X dsDNA High Sensitivity (HS) and Broad Range (BR) Assays.

The DeNovix Fluorometer utilizes a proprietary optical core and a versatile set of four fluorescence channels for excitation and emission detection of fluorophores. These channels enable fluorescent quantification of dsDNA, as well as ssDNA, RNA, and protein. The DeNovix Fluorometer is pre-configured for these assays and automatically uses the correct excitation and emission filters.

dsDNA Quantification Assays

The Qubit™ 1X dsDNA HS Assay enables selective quantitation in the detection range of 0.1 ng – 120 ng total mass of dsDNA when using 1 – 20 µL in 200 µL assay volume. This equates to measuring dsDNA initial sample concentrations of 5 pg/µL – 120 ng/µL when using 1 – 20 µL of sample in 200 µL assay volume.

The standard detection range of the Qubit™ 1X dsDNA BR assay is 4 – 4000 ng total mass when using 1 – 20 µL in 200 µL assay volume. This equates to measuring dsDNA initial sample concentrations of 0.2 ng/µL – 4000 ng/µL when using 1 – 20 µL of sample in 200 µL assay volume.

Materials and Methods

The Qubit™ 1X dsDNA HS Assay Kit (Thermo Fisher Scientific cat #Q33230) and the Qubit™ 1X dsDNA BR Assay Kit (Thermo Fisher Scientific cat #Q33265) were used to perform both assays. Each kit has a mix-and-measure protocol and includes a working solution and two standards. Samples were measured in thin-walled, clear UV-transparent 0.5 mL PCR tubes (DeNovix cat #TUBE-PCR-0.5-500). 

Performance data for Qubit™ 1X dsDNA Quantification Assays were obtained on a Thermo Fisher Qubit™ 3.0 and a DeNovix QFX Fluorometer. Each assay was prepared as described in the manufacturer’s protocol. Samples were mixed and incubated at room temperature for 5 minutes. Three replicate measurements were taken for each sample.

A series of dilutions was gravimetrically prepared in the TE buffer using the Qubit™ HS and BR Assay standards. The samples for the HS assay were prepared from the 10 ng/µL standard, and the samples for the BR assay were diluted from the 100 ng/µL standard. Each sample was prepared to fall within the total mass limits of the respective assay. For each assay, 1 – 20 µL of each prepared sample was added to 180 – 190 µL working solution.The gravimetrically determined initial sample concentrations were between 0.009 and 9.9 ng/µL for the HS assay and between 0.19 and 99 ng/µL for the BR assay.

Performance Data

Table 1 shows data for the Qubit™ 1X dsDNA HS Assay measured on the QFX and the Qubit™ 3.0. Table 2 shows data for the Qubit™ 1X dsDNA BR Assay. Expected concentrations were calculated based upon the gravimetric dilutions.

Table 1: Qubit™ 1X dsDNA HS Assay Performance Data

Summary

The data presented in this technical document shows that the DeNovix QFX Fluorometer enables sensitive quantitation of dsDNA when using a Qubit™ 1X dsDNA Assay. The DeNovix QFX dsDNA quantitation performance is equivalent to Qubit™ performance when measuring dsDNA using both the Qubit™ 1X dsDNA HS and BR Assays.