Sep 05, 2025

Public workspaceDNA Quantification using the Qubit Fluorometer

 Forked from DNA quantification using the Quantus fluorometer
DOI
https://dx.doi.org/10.17504/protocols.io.261gekdeyg47/v1
  • Josh Quick1
  • 1University of Birmingham
  • ARTIC
Mia Weaver
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DOI: https://dx.doi.org/10.17504/protocols.io.261gekdeyg47/v1
External link: https://doi.org/10.1016/j.remle.2020.05.007
Protocol Citation: Josh Quick 2025. DNA Quantification using the Qubit Fluorometer. protocols.io https://dx.doi.org/10.17504/protocols.io.261gekdeyg47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 18, 2025
Last Modified: September 05, 2025
Protocol Integer ID: 224878
Keywords: DNA, DNA Quantification, Qubit, Fluorometer, dsDNA HS Assay, dsDNA, assay kit for dsdna quantification, qubittm dsdna h, dsdna quantification, dna quantification, qubit fluorometer step by step guide, qubit fluorometer step, dna, assay kit
Abstract
Step by step guide to utilising the QubitTM dsDNA HS (High Sensitivity) Assay Kit for dsDNA quantification.
Guidelines
This protocol utilises the QubitTM dsDNA HS assay on the QubitTM 4 Fluorometer. The QubitTM 3.0 Fluorometer is also compatible with this protocol. If utilising the QubitTM Flex, the protocol will differ slightly in reading the standards and samples, and the respective tubes utilised, but the same working solution can be made.
Materials
Equipment:
EquipmentSupplierCatalogue Number
Qubit™ 4 FluorometerThermoFisher ScientificQ33226
Vortex
Microcentrifuge


Consumables and Reagents:
Consumables/ReagentSupplierCatalogue Number
Qubit™ dsDNA Quantification AssayThermoFisher ScientificQ32851/Q32854
Qubit™ Assay TubesThermoFisher ScientificQ32856


Troubleshooting
DNA Quantification using the Qubit Fluorometer
Remove the QubitTM Standards from the fridge and allow to come to room temperature.
Note
The QubitTM dsDNA HS Assay delivers optimal performance when all solutions are at room temperature (Temperature22 °C – Temperature28 °C ).


Prepare the QubitTM working solution in a fresh tube by diluting the QubitTM dsDNA HS Reagent 1:200 in QubitTM dsDNA HS Buffer.
Note
Each standard tube requires Amount190 µL of QubitTM working solution, and each sample tube
requires Amount199 µL . Prepare sufficient QubitTM working solution to accommodate all
standards and samples.

Note
QubitTM dsDNA HS Buffer and QubitTM dsDNA HS Reagent can be stored at the
room temperature but the QubitTM dsDNA HS Reagent should be protected from light.


Set up two QubitTM Amount0.5 mL tubes for calibration, labelling them 'Standard #1' and 'Standard #2' on the lid.
Note
Avoid labelling the tubes on the side as this may interfere with the instrument readings.

Add Amount190 µL of QubitTM working solution to each of the tubes used for standards.
Add Amount10 µL of each QubitTM standard to the appropriate tube.
Note
Ensure the QubitTM standards have been sufficiently vortexed before use.

Add Amount199 µL QubitTM working solution to the a new QubitTM sample tube and add Amount1 µL of your sample to the appropriate tube.
Vortex all QubitTM tubes for Duration00:00:03 , then spin them down. Allow all tubes to incubate at room temperature for Duration00:02:00 .
On the Home screen of the QubitTM Fluorometer select the dsDNA High Sensitivity as the assay type. Next, calibrate the instrument by reading the standards.
Insert the tube containing Standard #1 into the sample chamber, close the lid, and then press Read standard. When the reading is complete, remove Standard #1. The instrument displays the results on the Read standard screen.
Repeat the previous step but utilise Standard #2.
Once calibrated, any samples utilising the calibrated QubitTM working solution can be ran. Select the sample volume (Amount1 µL ) and units (ng/μL). Insert a sample tube into the sample chamber, close the lid, and then press Read tube.
When the reading is complete (~Duration00:00:03 ) remove the sample tube. The instrument displays the results on the assay screen. The top value is the concentration of the original sample. The bottom value is the dilution concentration.
Repeat for any remaining samples.
Protocol references
This protocol utilises the QubitTM dsDNA HS Assay Kit User Guide supplied by ThermoFisher Scientific (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/Qubit_dsDNA_HS_Assay_UG.pdf).