Qubit 4 Fluorometer

SOP-L213 Revision 0.1
Author

Marc Los Huertos

EA Program, Pomona College

Published

2026-06-26

1 Purpose

This procedure describes the operation, calibration, and maintenance of the Invitrogen Qubit 4 Fluorometer. The Qubit uses fluorescent dyes that bind selectively to the target molecule (dsDNA, ssDNA, RNA, or protein), providing more accurate quantification than UV absorbance methods, especially at low concentrations or when contaminants are present. For the role of Qubit measurements within the DNA extraction workflow, see SOP-L700 DNA Extraction.

2 Scope

  • Applies to: All use of the Qubit 4 Fluorometer in the EA Program laboratory, including dsDNA Broad Range (BR), dsDNA High Sensitivity (HS), RNA, and protein assays
  • Does not apply to: UV-Vis spectrophotometric quantification (see SOP-L212), Turner Designs fluorometry for chlorophyll or other environmental analytes (see SOP-L203)
  • Location: Instrument bench, SGM 133

3 Responsibilities

Role Responsibility
Lab PI Approve SOP, authorize instrument access
Lab Manager Maintain reagent inventory, schedule service
Trained Personnel Follow this SOP, report malfunctions
New Students Complete training before independent use

4 Definitions

  • Broad Range (BR) assay: A Qubit assay kit designed for DNA concentrations of 2–1,000 ng/µL; less sensitive than the HS assay but covers a wider dynamic range; used when high DNA yield is expected.
  • dsDNA: Double-stranded DNA; the form quantified by most Qubit DNA assay kits; the fluorescent dye binds specifically to dsDNA, not to ssDNA, RNA, or protein contaminants.
  • Fluorophore: A fluorescent chemical compound that absorbs light at a specific excitation wavelength and emits light at a longer emission wavelength; the Qubit dye is a fluorophore that binds selectively to dsDNA, ssDNA, or RNA depending on the kit.
  • High Sensitivity (HS) assay: A Qubit assay kit designed for DNA concentrations of 0.2–100 ng/µL; more sensitive than BR; preferred for low-yield environmental DNA extractions from soil or water.
  • Qubit assay tube: A thin-walled 0.5 mL PCR tube in which the working solution and sample are mixed for measurement; must be free of fluorescent contaminants; do not substitute with other tube types.
  • Working solution: A freshly prepared mixture of Qubit reagent diluted in buffer at a ratio specified by the kit (usually 1:200); prepared fresh for each session; stable for up to 3 hours at room temperature.

5 Safety and Hazards

WarningHazards
  • Chemical: Qubit reagent concentrate contains DMSO. Avoid skin contact; review the SDS before handling.
  • Biological: Samples may contain hazardous biological material. Treat all samples as potentially infectious.

5.1 Required PPE

5.2 Emergency Procedures

For chemical splashes, flush with water for at least 15 minutes and consult the SDS. For biological spills, clean per SOP-L002 Laboratory Safety.

6 Equipment and Materials

6.1 Equipment

Item Location Notes
Qubit 4 Fluorometer Instrument bench, SGM 133 Invitrogen / Thermo Fisher
USB drive or Wi-Fi connection – For data export

6.2 Reagents and Consumables

Item Catalog No. Storage
Qubit dsDNA BR Assay Kit Q32850 Room temperature
Qubit dsDNA HS Assay Kit Q32851 Room temperature
Qubit assay tubes (0.5 mL, thin-wall) Q32856 Room temperature
Nuclease-free water Various Room temperature
Note

Use only the thin-wall 0.5 mL Qubit assay tubes (Cat. Q32856). Standard microcentrifuge tubes are not optically compatible and will produce inaccurate readings.

7 Procedure

7.1 Selecting the Assay

Choose the assay based on expected concentration (Invitrogen, 2019):

Assay Detection Range When to Use
dsDNA BR 2–1,000 ng Most soil/sediment extractions
dsDNA HS 0.2–100 ng Low-yield samples (e.g., water filters, dilute extracts)
RNA BR / HS See kit documentation RNA quantification
Protein See kit documentation Protein quantification

7.2 Preparing the Working Solution

  1. Calculate the volume of working solution needed: 200 uL per assay tube (all samples plus 2 standards)
  2. Dilute the Qubit reagent concentrate 1:200 in Qubit buffer. For example, for 10 tubes: 10 uL reagent + 1,990 uL buffer = 2,000 uL total.
  3. Vortex briefly to mix
Important

Prepare the working solution fresh for each session. Do not store diluted working solution.

7.3 Preparing Standards

  1. Label two Qubit assay tubes: Std 1 and Std 2
  2. Add 190 uL of working solution to each tube
  3. Add 10 uL of Standard 1 to the first tube and 10 uL of Standard 2 to the second tube
  4. Vortex each tube briefly (2–3 seconds)

7.4 Preparing Samples

  1. Label one Qubit assay tube per sample
  2. Add 198 uL of working solution to each tube
  3. Add 2 uL of sample (DNA extract, RNA, or protein solution) to the corresponding tube
  4. Vortex each tube briefly (2–3 seconds)
Tip

The sample volume can range from 1–20 uL as long as the total volume in each tube equals 200 uL (adjust the working solution volume accordingly). Using 2 uL of sample minimizes consumption of precious extracts.

7.5 Incubation

  1. Incubate all tubes (standards and samples) at room temperature for 2 minutes (Invitrogen, 2019)
  2. Do not incubate for longer than 30 minutes; fluorescence signal decays over time

7.6 Calibration

  1. On the Qubit home screen, select the appropriate assay (e.g., dsDNA Broad Range)
  2. Tap Read Standards
  3. Insert the Std 1 tube, close the lid, and tap Read
  4. When prompted, insert the Std 2 tube, close the lid, and tap Read
  5. Verify that the calibration is accepted (green checkmark). If it fails, prepare fresh standards and repeat.

7.7 Reading Samples

  1. Tap Run Samples
  2. Set the sample volume to match the volume of sample added (e.g., 2 uL)
  3. Set the output units (ng/uL is standard for DNA extracts)
  4. Insert the sample tube, close the lid, and tap Read
  5. Record the concentration (ng/uL, corrected for dilution by the instrument)
  6. Repeat for all samples

7.8 Data Export

  1. Export results via USB or Wi-Fi to the designated project folder
  2. Use a consistent file-naming convention: YYYY-MM-DD_Qubit_ProjectName.csv
  3. Record results in the lab notebook and/or project data sheet

7.9 Shutdown and Daily Maintenance

  1. Remove all tubes from the instrument
  2. Wipe the sample chamber with a lint-free tissue if any liquid was spilled
  3. Close the lid
  4. Dispose of used assay tubes (see Section 8)

8 Quality Control and Acceptance Criteria

  • Run fresh standards at the beginning of each session; do not rely on a previous calibration
  • Standards must be within the acceptable range indicated by the instrument (green checkmark)
  • If a sample reads “Out of Range,” re-prepare it using the other assay sensitivity (e.g., switch from BR to HS for low-concentration samples)
  • Acceptable DNA yield from 0.25 g of soil: typically 1–20 ng/uL in a 100 uL elution, depending on soil type
  • If the NanoDrop and Qubit concentrations disagree by more than a factor of two, the NanoDrop reading is likely inflated by contaminants; trust the Qubit value for downstream planning

TBD – establish a periodic performance check using Qubit DNA standards of known concentration.

9 Waste Disposal

Waste Type Disposal Method
Used Qubit assay tubes Standard trash (rinse if biohazardous)
Leftover working solution Standard drain (dilute DMSO, non-hazardous at this concentration)
Pipette tips Standard trash (or biohazard if samples are hazardous)

10 References

Invitrogen. (2019). Qubit 4 fluorometer user guide. Thermo Fisher Scientific. https://www.thermofisher.com/us/en/home/references/protocols/nucleic-acid-purification-and-analysis/qubit-assay-protocols.html

11 Training Record

Before using the Qubit independently, personnel must:

  1. Read this SOP in full
  2. Complete SOP-L002 Laboratory Safety training
  3. Observe a trained operator demonstrate standard preparation, calibration, sample reading, and data export
  4. Perform a practice session (calibration plus at least 5 samples) under supervision
  5. Be signed off by the PI or lab manager