Qubit™ 1X dsDNA HS Assay (High Sensitivity)

Safety & Handling

• Treat Component A (working solution containing dye) as a potential mutagenic reagent; avoid skin/eye contact and dispose according to local regulations. (p.2)
• Wear appropriate PPE (lab coat, gloves, eye protection).
• Light sensitivity: Protect assay tubes from light; signal in prepared tubes is stable for ≤3 hours at room temperature when protected. (p.1)

Reagents, Consumables & Equipment

Kit (Q33230 – 100 assays; Q33231 – 500 assays):

• Component A: Qubit™ 1X dsDNA HS Working Solution (ready to use) — store 2–8 °C, protect from light.
• Component B: Standard #1 (0 ng/µL in TE) — store 2–8 °C.
• Component C: Standard #2 (10 ng/µL in TE) — store 2–8 °C. When stored as directed, kit stability is ≥6 months from receipt. (p.1, Table 1)

Not provided:

• Qubit™ Assay Tubes, thin‑wall clear 0.5 mL PCR tubes (Cat. Q32856) for Qubit™ 4 or Qubit™ Flex Tube Strips 8 × 200 µL (Cat. Q33252).
• Calibrated pipettes and a P‑2 for 1–2 µL additions; nuclease‑free tips. (pp.2–3)

Instruments:

• Qubit™ 4 or Qubit™ Flex Fluorometer. (Qubit™ 2/3 require the assay program download prior to use.) (p.1)

Critical Parameters & Environmental Conditions

• Assay temperature: Run at room temperature (18–28 °C). Avoid warming tubes by hand or prolonged instrument residence; the fluorometer can heat solutions over minutes.
• Incubation: After mixing standards or samples with working solution, incubate 2 minutes before reading. Signal remains stable ≤3 hours (light‑protected).
• Photostability & re‑reads: Fluorescence drop is <0.3% after 9 reads and <2.5% after 40 reads, but temperature rise inside the instrument can temporarily reduce signal. If re‑reading the same tube, remove it and equilibrate ~30 s at room temperature before the next read.

Preparation (Before You Begin)

1. Equilibrate kit components to room temperature.
2. Determine whether you will calibrate now or reuse the last calibration. For new users, perform a fresh calibration each run; experienced users may reuse the stored calibration, considering temperature/pipetting conditions. Note the stability limit of prepared standards/samples (≤3 h).
3. Plan total working solution required: each tube is 200 µL final volume. Standards require 190 µL working solution + 10 µL standard; samples require (200 µL – sample volume) working solution.

Quick volume guide (per tube): Sample 1 µL → 199 µL working solution; 2 µL → 198 µL; 5 µL → 195 µL; 10 µL → 190 µL; 20 µL → 180 µL. Final volume must be 200 µL.

Tube setup & labeling

1. Set out two tubes for standards and one tube per sample (or one strip position per sample for Flex). Use the tube type appropriate for your instrument.
2. Label tube lids (not the sides) to avoid interfering with the read. For standards, ensure correct order for calibration.

Load standards and samples

3. Add 10 µL of Standard #1 to the “Std1” tube and 10 µL of Standard #2 to the “Std2” tube.
4. Add 1–20 µL of each sample to its labeled tube (use a P‑2 for 1–2 µL).
5. Add Qubit™ 1X dsDNA HS Working Solution to each tube to reach 200 µL total volume. (Standards: 190 µL; samples: 180–199 µL; consider pre‑aliquoting working solution to avoid cross‑contamination.)
6. Vortex 3–5 s to mix. Incubate 2 min at room temperature, protected from light.

Instrument reading

A) Qubit™ Flex Fluorometer 7. Home → 1X dsDNA HS → Read Standards & run samples. 8. Read Standard #1, then Standard #2 (~3 s each). Review the standard graph, then Next. 9. On Insert samples, select active positions; choose units (e.g., ng/µL). Optionally enter kit lot, Tags, Sample IDs. 10. On Sample volume, enter 1–20 µL used per tube (affects assay accuracy range). 11. Insert sample strip, Run samples (~3 s). Results list shows original sample concentration. Add more samples as needed. Optional Molarity/Normalization calculators are available.

B) Qubit™ 4 Fluorometer 7. Home → DNA → 1X dsDNA HS → Read Standards. 8. Read Standard #1, then Standard #2 (~3 s each), review, then Run samples. 9. Select sample volume (1–20 µL) and units on the assay screen.
10. Insert each sample tube and Read tube (~3 s). The top value is original sample concentration; the bottom value is the dilution concentration. Repeat for all samples.

Calibration notes: The instrument fits a curve (modified Hill plot) between the two standards; you may run a new calibration or reuse the previous one (experienced users).

Calculations & Reporting

• The fluorometer automatically converts fluorescence to concentration via the two‑point calibration curve; report the “original sample concentration” produced by the instrument in the selected units.
• Record for each sample: sample ID, input volume, final total volume (200 µL), instrument model/serial, calibration used (new/previous), kit lot number, ambient temperature (if recorded), read time, and result.

Assay Performance, Selectivity & Tolerances

• Selectivity: Highly selective for dsDNA over RNA

• Contaminant tolerance: Results typically vary by <10% in the presence of many common contaminants at indicated final assay concentrations—for example:

  • NaCl 50 mM (≈1 M in a 10 µL sample), MgCl₂ 1 mM, Sodium acetate 30 mM, Ethanol 1%, Phenol 0.1%, SDS 0.002%, as well as RNA, ssDNA, proteins (BSA/IgG), and dNTPs at levels ~equal to the dsDNA concentration. For best results, add the same contaminant amount to the standards.

• Dynamic range & confidence: Core detection 0.2–100 ng total DNA per tube with recommended input concentrations 10 pg/µL to 100 ng/µL.

Quality Control

• Standards must read in correct order and generate a plausible calibration curve; if not, repeat calibration with fresh standards.
• Use consistent sample volumes (1–20 µL) across a batch when possible, as this affects the assay accuracy window.
• Avoid temperature drift: do not hold tubes in hand; minimize time in instrument; if re‑reading, equilibrate 30 s at room temperature.
• Document kit lot, instrument, calibration, and incubation time (≥2 min) in the run log.

Limitations

• If sample concentration is outside the HS assay’s accurate range, consider other Qubit™ assays (e.g., dsDNA BR, ssDNA, RNA kits).
• Maximum stability of prepared tubes is 3 h at room temperature when protected from light; read within this window.

Troubleshooting (quick guide)

• Low/variable readings: Verify correct total volume (200 µL), adequate mixing (vortex 3–5 s), and 2‑minute incubation; re‑run calibration; check temperature handling practices.
• Standards fail or curve looks wrong: Replace standards, ensure correct order and volumes; avoid labeling tube sides; recalibrate.
• Signal drift on repeated reads: Remove tube between reads and allow ~30 s equilibration.

Waste Disposal

Dispose of used tips/tubes and any remaining dye‑containing working solution per institutional chemical waste guidelines (treat dye as potentially mutagenic).

References

• **Qubit™ 1X dsDNA HS Assay Kits – User Guide, Pub. No. MAN0017455 Rev. C.0 (08 Dec 2020).