Qubit dsDNA High Sensitivity Quantification Protocol
Protocol Overview
This protocol uses fluorometry to accurately measure double-stranded DNA concentration in mosquito extracts. Qubit dyes specifically bind dsDNA and fluoresce, ignoring contaminants like RNA, proteins, and salts. This is the gold standard for DNA quantification before PCR setup.
Detection range: 0.005-120 ng/µL (High Sensitivity Kit)
Sample volume: 1-2 µL
Time required: 10-15 minutes for 8 samples
Materials and Equipment
Instrumentation
- Qubit Flex Fluorometer (or Qubit 4, Qubit 3.0)
- P2 and P200 pipettes (calibrated)
- Vortex mixer
- Mini centrifuge (for quick spin)
Reagents (BioDynami dsDNA HS Kit or Invitrogen Qubit dsDNA HS Kit)
- Qubit dsDNA HS Reagent (fluorescent dye in DMSO)
- Qubit dsDNA HS Buffer
- Qubit dsDNA HS Standard #1 (0 ng/µL - blank)
- Qubit dsDNA HS Standard #2 (100 ng/µL)
- Qubit assay tubes (0.5 mL thin-walled, optically clear)
Safety Equipment
- Lab coat
- Safety glasses
- Nitrile gloves
Important Safety Notes
- Qubit dye contains DMSO (dimethyl sulfoxide) - wear gloves, DMSO penetrates skin
- Do not expose working solution to bright light for >30 min (dye photobleaches)
- Use only thin-walled Qubit tubes - thick PCR tubes will give incorrect readings
Part 1: Calculations and Reagent Preparation
Step 1: Calculate Working Solution Volume
Formula
Number of samples + 2 standards + 1 extra = Total tubes
Working solution per tube: 199 µL (for 1 µL sample) or 198 µL (for 2 µL sample)
Total working solution = Total tubes × 199 µL
1 Count your DNA samples (example: 8 samples)
8 samples + 2 standards + 1 extra = 11 tubes
Working solution needed: 11 × 199 µL = 2,189 µL
Round up to: 2,200 µL
Step 2: Prepare Qubit Working Solution
2 Calculate reagent volumes:
Total working solution: 2,200 µL
Dilution ratio: 1:200 (dye:buffer)
Qubit dye reagent: 2,200 ÷ 200 = 11 µL
Qubit HS buffer: 2,200 - 11 = 2,189 µL
3 In a 15 mL conical tube, mix:
- 11 µL Qubit dsDNA HS Reagent (use P20 pipette)
- 2,189 µL Qubit dsDNA HS Buffer (use P1000 pipette, 2× transfers)
4 Vortex working solution for 5 seconds to mix thoroughly
Pro Tip
Working solution is stable for 4 hours at room temperature if protected from light. For longer storage, wrap tube in aluminum foil. Do not freeze.
Part 2: Prepare Standards (Calibration Curve)
Step 3: Set Up Standard Tubes
1 Label two Qubit assay tubes:
- S1 (Standard 1 - 0 ng/µL blank)
- S2 (Standard 2 - 100 ng/µL)
2 Add to each standard tube:
- 190 µL working solution (P200 pipette)
3 Add standards:
- To S1 tube: 10 µL of Standard #1 (0 ng/µL)
- To S2 tube: 10 µL of Standard #2 (100 ng/µL)
Critical
Use fresh pipette tips for each standard to avoid cross-contamination. Never re-use standard tubes from previous assays.
4 Cap tubes, vortex 3 seconds, quick spin to collect liquid at bottom
Part 3: Prepare DNA Samples
Step 4: Set Up Sample Tubes
1 Label Qubit assay tubes with sample IDs
2 Add to each sample tube:
- 199 µL working solution (P200 pipette)
Sample Volume Options
Standard protocol (1 µL sample):
- 199 µL working solution + 1 µL DNA = 200× dilution
- Best for most mosquito DNA extracts (10-50 ng/µL)
Low concentration samples (2 µL sample):
- 198 µL working solution + 2 µL DNA = 100× dilution
- Use if DNA concentration expected <5 ng/µL
- Improves accuracy for dilute samples
3 Before pipetting each DNA sample:
- Vortex DNA stock tube for 5 seconds
- Quick spin to collect liquid at bottom
- Pipette from middle of liquid (avoid settled DNA at bottom or condensation at top)
4 Add DNA to sample tubes:
- 1 µL DNA sample (P2 pipette, set to 1.0 µL)
- Mix by pipetting up and down 2-3 times
Pipetting Accuracy is Critical
Qubit readings are only as accurate as your pipetting. For 1 µL volumes:
- Use a calibrated P2 pipette (NOT P10 or P20)
- Set to exactly 1.0 µL
- Check for air bubbles in tip after aspiration
- Dispense slowly to ensure complete delivery
5 Cap each tube, vortex 3 seconds, quick spin
Part 4: Incubation
Step 5: Allow Dye-DNA Binding Equilibrium
1 Incubate all tubes (standards + samples) at room temperature for 2 minutes
Why Incubate?
Dye molecules need time to intercalate between DNA base pairs. Binding equilibrium is reached in ~2 minutes. Measuring earlier gives falsely low readings. Measuring later (up to 30 min) is acceptable.
2 During incubation, prepare Qubit instrument (see Part 5)
Part 5: Qubit Flex Measurement
Step 6: Power On and Select Assay
1 Power on Qubit Flex (button on front panel)
2 On touchscreen, select "dsDNA" assay type
3 Select "High Sensitivity" (HS) range
4 Choose "Read Standards"
Step 7: Calibrate with Standards
5 When prompted, insert Standard 1 tube into Qubit
6 Close lid, press "Read"
7 Wait 3-5 seconds for measurement
8 Remove Standard 1 tube
9 Insert Standard 2 tube
10 Close lid, press "Read"
11 Instrument displays "Calibration successful" if standards pass QC
Calibration Failure
If Qubit rejects calibration:
- Standards may be contaminated - prepare fresh standards
- Wrong tube type - use only thin-walled Qubit tubes
- Dye degraded - prepare fresh working solution
- Bubbles in tubes - tap tubes to remove, re-read
Step 8: Measure DNA Samples
12 Select "Read Samples"
13 For each sample:
- Insert sample tube into Qubit
- Enter sample ID on touchscreen
- Enter sample volume used (1 µL or 2 µL)
- Close lid, press "Read"
- Wait 3-5 seconds
- Record concentration displayed (ng/µL) - instrument auto-corrects for dilution
- Remove tube
Pro Tip
Qubit automatically calculates original DNA concentration by multiplying measured fluorescence by dilution factor (200× or 100×). You do not need to do manual calculations unless recording raw fluorescence values.
Step 9: Record Data
14 For each sample, record in lab notebook:
- Sample ID
- Qubit reading (ng/µL)
- Sample volume used (1 µL or 2 µL)
- Date and instrument ID
15 Optional: Export data to USB drive (Qubit Flex feature)
Part 6: Interpreting Results
Typical Results for Mosquito DNA
| Reading (ng/µL) |
Quality |
PCR Use |
| 50-120 |
Excellent yield |
Dilute to 20 ng/µL |
| 10-50 |
Optimal for PCR |
Use 1 µL in PCR |
| 2-10 |
Low but usable |
Use 3-5 µL in PCR |
| 0.2-2 |
Very low |
Use 5 µL max |
| <0.2 |
Insufficient |
Re-extract or use 10 µL |
| "Too low" |
Below detection |
Re-measure with 2 µL |
| "Too high" |
Above detection |
Dilute 1:10 and re-measure |
Part 7: PCR Template Calculations
Scenario 1: DNA in Optimal Range
Example: Qubit reads 25 ng/µL
PCR setup:
- Target: 25 ng DNA per 25 µL reaction
- Template volume: 25 ng ÷ 25 ng/µL = 1 µL
- Add 1 µL DNA directly to PCR master mix
Scenario 2: DNA Too Concentrated
Example: Qubit reads 85 ng/µL
Option A - Dilute to working stock:
Goal: Make 30 µL at 20 ng/µL
C₁V₁ = C₂V₂
85 × V₁ = 20 × 30
V₁ = 7.1 µL
Mix: 7.1 µL DNA + 22.9 µL elution buffer = 30 µL at 20 ng/µL
Use 1 µL of diluted stock in PCR
Option B - Use less volume directly:
Template volume = 25 ng ÷ 85 ng/µL = 0.29 µL
Round to 0.5 µL (difficult to pipette accurately with P2)
Better to dilute (Option A)
Scenario 3: DNA Too Dilute
Example: Qubit reads 4 ng/µL
Use more template volume:
Goal: 25 ng total in PCR
Template volume = 25 ng ÷ 4 ng/µL = 6.25 µL
Round to 6 µL
PCR recipe:
- 12.5 µL Q5 Master Mix
- 6.0 µL DNA template (instead of 1 µL)
- 1.0 µL forward primer
- 1.0 µL reverse primer
- 4.5 µL water (instead of 9.5 µL)
Total: 25 µL
Part 8: Troubleshooting
Problem: "Sample concentration too low to read"
Causes:
- DNA concentration <0.005 ng/µL (below HS range)
- Forgot to add DNA sample
- DNA not mixed before pipetting (settled at bottom of tube)
Solutions:
- Vortex DNA stock thoroughly, re-prepare sample with 2 µL instead of 1 µL
- Check original tube - is there visible liquid?
- If still "too low," DNA extraction failed - re-extract tissue
Problem: "Sample concentration too high to read"
Causes:
- DNA concentration >120 ng/µL (above HS range)
- Excellent extraction of high molecular weight DNA
- Pipetting error (added 5 µL instead of 1 µL)
Solutions:
- Dilute DNA 1:10 with elution buffer
- Re-measure diluted sample
- Multiply result by 10 to get original concentration
Problem: Duplicate Readings Differ by >10%
Causes:
- DNA not homogeneous (not vortexed before pipetting)
- Bubbles in tube interfering with fluorescence
- Pipetting error (inconsistent volumes)
Solutions:
- Vortex DNA stock 10 seconds, let settle 30 seconds, pipette from middle
- Tap tubes to remove bubbles before reading
- Use calibrated pipette; practice with water first
Problem: Qubit Reads Much Lower Than NanoDrop
This is NORMAL for mosquito DNA
Explanation:
- NanoDrop measures all UV-absorbing molecules (DNA + RNA + proteins + melanin)
- Qubit measures only dsDNA
- Mosquito extracts contain melanin (eye pigments) that absorb UV but don't bind Qubit dye
Action: Trust Qubit for PCR calculations. This is why Qubit is the gold standard.
Part 9: Post-Protocol Cleanup
Step 10: Discard Used Tubes
1 Qubit reagent waste can be discarded in regular trash (dye is non-toxic)
2 Discard used Qubit tubes (not reusable)
3 Store leftover DNA samples at -20°C
Step 11: Store Reagents
4 Return Qubit dye reagent to 4°C (refrigerator)
5 Store buffer at room temperature
6 Store standards at 4°C
Reagent Shelf Life
- Unopened kit: 12 months at 4°C
- Opened dye reagent: 6 months at 4°C (protected from light)
- Working solution: Use within 4 hours (or store wrapped in foil)
- Standards: Single use only; do not re-use
Data Recording Template
| Sample ID |
Sample Volume (µL) |
Qubit Reading (ng/µL) |
Total Yield (ng)* |
PCR Template Volume |
| Standard 1 |
10 |
0.0 |
— |
— |
| Standard 2 |
10 |
~100 |
— |
— |
| Sample 1 |
1 |
_______ |
_______ |
_______ |
| Sample 2 |
1 |
_______ |
_______ |
_______ |
| Sample 3 |
1 |
_______ |
_______ |
_______ |
*Total Yield (ng) = Concentration (ng/µL) × Elution Volume (µL)
Example: 25 ng/µL × 30 µL elution = 750 ng total
Key Takeaways
Critical Concepts
- Qubit is dsDNA-specific: Ignores RNA, proteins, salts, melanin
- Accuracy depends on pipetting: Use calibrated P2 for 1 µL volumes
- Vortex before every pipetting: DNA settles quickly
- Trust Qubit over NanoDrop: For PCR calculations, always use Qubit concentration
- Standards must pass QC: If calibration fails, prepare fresh reagents
- 2-minute incubation is required: Dye-DNA binding needs equilibrium time
Best Practice
Use Qubit and NanoDrop together:
- Qubit: Accurate dsDNA concentration for PCR setup
- NanoDrop: Purity ratios (A260/A280, A260/A230) for troubleshooting
- If NanoDrop >> Qubit: Contaminants present, but trust Qubit
- If ratios poor: Dilute DNA for PCR or add BSA to reaction