Fluorometric dsDNA Quantification (Qubit dsDNA HS Assay)
Fluorometric measurement of double-stranded DNA concentration using the Qubit dsDNA HS (High-Sensitivity) assay. Covers the 0.01-100 ng/µL range with ±10% typical accuracy — sensitive enough to measure even low-yield extracts from small or inhibitor-heavy samples (pollen pellets, dried insects). Primary QC readout for post-extraction feasibility decisions in the Stage 0.5 POC, and for ONT library prep input mass gating (RBK114 requires 50-400 ng per barcode). Written for a novice audience.
Version History
Version 0.1.2 Latest locked
Effective: 2026-05-21F12.v2 declared outputs: DNA concentration (NCIT:C179798) in ng/µL (UO:0010050). Bridge change; protocol body, parameters, equipment, materials, references identical to v0.1.1. First F12.v2-shape bump on garage-genomics side — declaration-driven matrix will auto-populate this column on any new Process against this version.
Version 0.1.1
Effective: 2026-05-18Tagged 5 process-scoped parameters with scope='process'; sample-scoped params (sample_volume_ul, working_solution_volume_ul) keep scope='sample'. Measurement-category fixture for M3 matrix-inheritance arc — exercises the F7-closing path (sample-scoped capture at matrix surface). Bridge change only; protocol body identical to v0.1.0.
Version 0.1.0 Viewing locked
Effective: 2026-04-21Initial release. Standard Qubit dsDNA HS fluorometric quantification protocol. Atomic measurement technique supporting the Stage 0.5 POC (readout for procedure 82 and 83 extracts) and general-purpose DNA input QC for library prep and PCR normalisation. Chemistry is the manufacturer-specified procedure — adapted for a novice audience.
Procedure Details
- BSL-1 work only.
- Qubit working dye — proprietary Invitrogen dye; mildly cytotoxic; handle with gloves. Manufacturer MSDS is the authoritative safety reference.
- Qubit assay tubes — use only the manufacturer's thin-wall clear Qubit tubes. Standard microcentrifuge tubes have too much fluorescence background and will give nonsense readings.
- Pipetting hygiene. Filter tips for sample transfer into the assay tube to prevent cross-contamination between extracts — the assay will read any dsDNA contamination faithfully and cumulatively.
- Not a clinical measurement. Research-grade quantification; not validated for clinical or diagnostic use.
- Instrument calibration drift. Standards should be re-read at the start of each session. Older kit lots drift fastest.
Qubit working solution (prepare fresh for each batch):
- 199 parts Qubit dsDNA HS buffer + 1 part Qubit dsDNA HS dye (1:200 dilution).
- Store in a light-protected container (the kit's plastic reservoir, or an amber tube wrapped in foil).
- Stable for ~3 hours at room temperature, ~24 hours at 4°C. Light-sensitive — prepare, cover, use promptly.
- Volume calculation: (N_samples + N_standards) × 200 µL = total working solution volume needed. For 10 samples + 2 standards: 12 × 200 = 2.4 mL of working solution.
Qubit standards (2 tubes per kit session):
- Standard #1 = 0 ng/µL (blank, pure buffer with dye)
- Standard #2 = 10 ng/µL (dsDNA at known concentration for calibration)
- Both are provided in the kit. Fresh reading required at each session unless the kit lot is used within 1-2 days of last calibration.
Sample specification:
- Input volume: 1-20 µL per sample (typically 2 µL).
- If sample is expected above 100 ng/µL, either reduce input volume (e.g., 1 µL) or switch to Qubit dsDNA BR (Broad Range) kit — HS dye saturates above its range and returns inaccurate (low-biased) reads.
Mental model. The Qubit dsDNA HS dye fluoresces only when bound to double-stranded DNA. Single-stranded DNA, RNA, protein, and most contaminants don't interact — which is why the Qubit reads DNA mass selectively even in messy extracts. The fluorometer is calibrated with the two standards at the start of each session, and sample reads are interpolated on that 2-point standard curve.
Why this matters for the POC. Every extract from procedures 81 and 82 needs a Qubit read as its pass/fail gate:
- Fail gate: < 1 ng/µL on a 50 µL resuspension = < 50 ng total → not enough DNA for ONT library prep under any barcode plexity.
- Marginal gate: 1-4 ng/µL × 50 µL = 50-200 ng → usable for ONT at low plexity (24-barcode kit) or for amplicon PCR.
- Pass gate: > 4 ng/µL × 50 µL = > 200 ng → supports 96-plex RBK114 library prep with margin.
What Qubit doesn't tell you. Fragment size. An extract can read 100 ng/µL and still be completely sheared (< 500 bp fragments) — run it on procedure 73 agarose gel alongside the Qubit read for any ONT library prep decision.
- Per reading: 5-10 min (setup + 2 min incubation + read).
- Per batch: 15-30 min for up to 12 samples with 2 standards. Re-reading standards between batches adds 2 min.
- Fluorometer warm-up: 5-10 min from cold start. Keep the instrument warm between sessions if possible.
-
Microcentrifuge
Centrifugation
Specs: Brief-spin capable (no rotor specs critical)
Collect liquid at tube bottom before reading -
Micropipette
Liquid handling
Specs: P2 / P20 / P200; filter tips for extract handling
Dedicated set for post-extraction QC preferred -
Fluorometer
Measurement
Specs: Qubit 4 or equivalent (excitation ~485 nm, emission ~530 nm); HS-compatible firmware
Manufacturer-specified; other fluorometers may work but require custom calibration -
Vortex mixer
Mixing
Specs: Standard benchtop
Brief (2-3 sec) vortex only — extended vortex shears HMW DNA in the sample
-
Filter pipette tips
Consumable
Qty: 3 tips
Required for sample handling to prevent extract-to-extract cross-contamination — assay reads dsDNA faithfully and cumulatively -
Microcentrifuge tube
Consumable
Qty: 1 tube
For preparing bulk working solution — light-protected if possible -
Pipette tips
Consumable
Qty: 4 tips
Generic tips for working-solution and standards handling (non-extract contact) -
Qubit 1X dsDNA HS Assay Kit
(Invitrogen)
Q33231Fluorometric assay kit
Qty: 1 kit
Invitrogen Q33231. Contains working-solution dye, buffer, and 2-point calibration standards. ~500 reactions per kit.
Protocol Parameters Captured per-assay on each run; exported as ISA-Tab Parameter Value columns
| Name | Type | Required | Default | Unit | Description |
|---|---|---|---|---|---|
sample_volume_ul |
number |
2
|
— | Volume of DNA extract added to the assay tube, in µL. 2 µL standard; up to 20 µL acceptable for low-concentration extracts (working solution volume reduced accordingly to keep total 200 µL). | |
working_solution_volume_ul |
number |
198
|
— | Volume of Qubit working solution (dye + buffer 1:200) in the assay tube, in µL. Standard = 200 µL total - sample volume = 198 µL when sample is 2 µL. | |
incubation_time_min |
number | — |
2
|
minute (UO:0000031) | Dye-binding equilibration time before reading, in minutes. 2 min standard; up to 10 min acceptable; shorter reads are unreliable. |
read_replicate_count |
number | — |
1
|
— | Number of replicate reads per sample. 1 standard; 2-3 recommended for samples near the edge of detection (<0.1 ng/µL) or for any sample that will drive a high-value downstream decision. |
assay_kit_lot |
text | — | — | — | Kit lot number recorded for traceability. Required for any sample contributing to a published result; optional for internal QC screening. |
standards_read_this_session |
boolean |
true
|
— | Whether calibration standards were read this session. Always TRUE at the start of a fresh session; may be FALSE for subsequent samples within the same session if standards haven't drifted meaningfully. | |
assay_variant |
text | — |
HS
|
— | Qubit assay variant used. Valid values: HS (High Sensitivity, 0.01-100 ng/µL), BR (Broad Range, 0.2-1000 ng/µL). Use HS for most POC extracts; switch to BR for samples known to be > 50 ng/µL. |
Procedure Steps (Version 0.1.0)
Turn on the fluorometer and allow 5-10 min warm-up from cold. Pull the Qubit dsDNA HS Assay Kit from 4°C storage and allow dye + buffer to equilibrate to room temperature (5 min).
Calculate total Qubit working solution volume needed: (N_samples + 2 standards) × 200 µL. Round up to the nearest 200 µL for pipetting convenience.
Prepare Qubit working solution in a labelled 1.5 mL microcentrifuge tube or the kit's light-protected plastic reservoir: 199 parts HS buffer + 1 part HS dye, mix by gentle inversion. Do not vortex — bubbles interfere with fluorescence.
Prepare the 2 calibration standards: label 2 fresh Qubit assay tubes as Standard 1 and Standard 2. Add 190 µL of working solution to each. Add 10 µL of Standard 1 (0 ng/µL) to the first tube and 10 µL of Standard 2 (10 ng/µL) to the second. Vortex each for 2-3 seconds, centrifuge briefly.
Prepare the sample readings: label one fresh Qubit assay tube per sample. Add 198 µL of working solution to each, then 2 µL of the DNA extract. For low-concentration samples, use up to 20 µL of extract with correspondingly less working solution (total must remain 200 µL). Vortex each sample tube for 2-3 seconds, centrifuge briefly.
Incubate all tubes (standards + samples) at room temperature for 2 minutes — this is the dye-binding equilibration time. Longer incubation is fine up to 10 min; shorter gives unreliable reads.
At the fluorometer, calibrate with Standard 1, then Standard 2 per the instrument's on-screen prompts. Verify the calibration curve looks reasonable (the Qubit 4 reports an R² — should be > 0.99). Reject and re-read if the curve is suspect.
Read each sample tube on the fluorometer. Record both the displayed concentration (ng/µL) and the sample volume entered (e.g., 2 µL), so the instrument correctly reports the in-tube concentration of the original extract.
For any sample that reads at the top of the HS range (near 100 ng/µL) or 'out of range — high', dilute 1:10 in TE buffer and re-read. Alternatively, switch to the Qubit dsDNA BR (Broad Range) kit and re-run.
Record the measurement as a Process in LibreBiotech: input sample = extract tube, output = measurement readings. Write annotations on the extract sample using slots dna_concentration_ng_ul, dna_total_mass_ng, and qubit_assay_kit_lot per the completion notes. The ISA-Tab Assay Table will project these annotations into factor-value columns at export time.
Dispose of used Qubit assay tubes per biological waste procedures. Working solution can be re-used within 3 hours at room temperature; discard after 24 h even if refrigerated.
Expected outcome. A numeric concentration reading in ng/µL (or equivalent from the instrument, often pg/µL for very low reads). Record both the instrument reading and the calculated extract total mass (concentration × resuspension volume) as annotations on the output extract sample in LibreBiotech.
Reading ranges (HS kit):
| Reading | Interpretation | Action |
|---|---|---|
| "Out of range — low" or < 0.01 ng/µL | Below detection | Sample likely genuinely empty; verify extraction; consider larger input for retry |
| 0.01-0.1 ng/µL | Low but detectable | May be salvageable for PCR amplicon work; unsuitable for ONT library prep |
| 0.1-1 ng/µL | Low | PCR-usable; ONT low-plex feasible if concentrated via speedvac or ethanol precipitation |
| 1-10 ng/µL | Typical POC range | PCR-usable; ONT 24-96 plex feasible depending on total volume |
| 10-100 ng/µL | Strong extract | ONT any plexity; dilute before PCR to avoid inhibitor carryover |
| "Out of range — high" or > 100 ng/µL | Above HS range | Dilute 1:10 in TE and re-read on HS, OR switch to BR kit for direct read |
Recording results. Create annotations on the extract sample:
slot=dna_concentration_ng_ul,value_num=<reading>,unit_term_id=<ng/µL CURIE>slot=dna_total_mass_ng,value_num=<reading × resuspension_volume>,unit_term_id=<ng CURIE>slot=qubit_assay_kit_lot,value_text=<kit lot number for traceability>
Storage. Qubit-measured samples are unchanged by the reading; return to −20°C (HMW) or 4°C (short-term) as before.
Troubleshooting.
| Symptom | Likely cause | Fix |
|---|---|---|
| Standard reads out of expected range at calibration | Stale working solution; kit lot expired; dye exposed to light | Remake working solution; check kit expiration; wrap in foil during handling |
| Sample reads much lower than expected | Degraded/sheared DNA (HS dye still responds but ssDNA regions don't bind); or concentration really is that low | Run on agarose gel to confirm fragment state; if intact, accept the reading |
| Sample reads much higher than expected | Contamination from prior sample (pipetting); or RNA carryover (rare — dye is dsDNA-selective but pinpoint) | Use filter tips; redo with fresh aliquot; RNase treatment if extract is very RNA-heavy |
| Inconsistent replicate reads | Insufficient mixing; sample/working-solution stratification in assay tube | Vortex assay tube 3-5 sec; always centrifuge briefly to collect liquid at bottom before reading |
| Standards drift session-to-session | Instrument calibration aging; kit oxidation | Re-read both standards every session; replace kit if standards still drift after fresh working solution |
| Negative reading (rare) | Assay tube fluorescence background subtraction issue | Use only the manufacturer's Qubit tubes; reject any standard-microcentrifuge-tube reads |
References
- Invitrogen / Thermo Fisher Scientific. Qubit dsDNA HS Assay Kit — product manual (Q33231). Manufacturer-authoritative protocol for fluorometric dsDNA quantification. Link protocol
- Singer VL, Jones LJ, Yue ST, Haugland RP (1997). Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation. Analytical Biochemistry 249(2):228-238. (Foundational paper for the picogreen-class dye chemistry the Qubit dye is built on). DOI paper
- LibreBiotech procedure 82 — CTAB DNA Extraction (Cross-Tissue Reference Protocol) — upstream procedure producing extracts that this measurement characterises. Link protocol
- LibreBiotech procedure 83 — SDS + Salt-Precipitation DNA Extraction (Consumer-Safe Cross-Tissue Protocol) — upstream procedure producing extracts that this measurement characterises. Link protocol
- LibreBiotech procedure 73 — Agarose Gel Electrophoresis — complementary QC for fragment-size assessment that Qubit cannot provide. Link protocol