Safety

• Wear lab coat, gloves, and eye protection.
• Handle DNA with care to avoid contamination; keep samples on ice unless specified otherwise.
• Use DNase-free, low-bind plastics and tips with aerosol barriers.
• Dispose of waste according to institutional biosafety and chemical hygiene rules.

Materials and Reagents

• DNA stock solution (record ID, concentration, buffer).
• Diluent: Nuclease‑free water or low‑EDTA TE (choose based on downstream assay).
• Calibrated pipettes and sterile, aerosol‑resistant tips.
• Microcentrifuge tubes or 96‑well plate (DNase‑free, low-bind).
• Vortex mixer and brief‑spin microcentrifuge.
• Ice rack.
• Labels/marker and lab notebook or electronic record.

Definitions & Formulae

• 1:10 dilution = 1 part DNA + 9 parts diluent (final concentration = 0.1× stock).
• Serial 1:10 over n steps gives 1:10ⁿ overall (10⁻ⁿ × stock).
• C₁V₁ = C₂V₂ → V₁ = (C₂ × V_total)/C₁.

Preparation

• Thaw DNA on ice; briefly spin to collect contents.
• Verify or measure DNA concentration if needed (prefer fluorometric methods for accuracy at low ng/µL).
• Plan the dilution factor, number of steps, and final volumes. Use volumes ≥10× your pipette’s systematic error (e.g., prefer 100 µL over 10 µL when feasible).

Procedure A — Single 1:10 Dilution (example volumes shown)

1. Label a DNase‑free tube with sample ID, “1:10”, date, and initials.
2. Add diluent: 90 µL.
3. Add DNA stock: 10 µL.
4. Mix by pipetting up/down 10× or quick vortex (1 s); brief spin to collect.
5. Record calculation, volumes, and resulting expected concentration (C₂ = 0.1 × C₁).

Alternatives (same 1:10 ratio):

• 20 µL DNA + 180 µL diluent (200 µL total)
• 5 µL DNA + 45 µL diluent (50 µL total; accuracy depends on pipette)

Procedure B — Serial 1:10 Dilution Series (tube‑to‑tube)

1. Plan & label tubes as: 10⁻¹, 10⁻², 10⁻³, … (or 1:10, 1:100, 1:1000). Include sample ID and date.
2. Pre‑aliquot diluent into each tube (e.g., 90 µL per tube for 100 µL steps).
3. Step 1 (10⁻¹): Add 10 µL of DNA stock to tube 10⁻¹. Mix thoroughly; brief spin.
4. Step 2 (10⁻²): Using a fresh tip, transfer 10 µL from tube 10⁻¹ to tube 10⁻². Mix; brief spin.

Overall factors:

• Tube 10⁻¹ = 0.1× stock; 10⁻² = 0.01×; 10⁻³ = 0.001×, etc.

5. Repeat stepwise through the final tube, changing tips each transfer.
6. Optional blank: Include a diluent‑only tube/well to monitor contamination.
7. Record all volumes and any deviations.

Procedure C — Serial 1:10 in a 96‑well Plate (for qPCR or assays)

1. Map wells (e.g., A1 = stock input, A2 = 10⁻¹, A3 = 10⁻², …).
2. Dispense diluent into target wells (e.g., 90 µL each).
3. Add stock to the first dilution well (A2): transfer 10 µL from stock (A1) → A2; mix by pipetting 10×.
4. Serial transfer: Move 10 µL from A2 → A3, mix; continue across row/column as planned.
5. Change tips every transfer and avoid touching well walls above liquid to reduce carryover.
6. Seal plate as required; briefly spin if compatible.

Examples & Calculations

• Example 1 (single 1:10): You need 100 µL at 1:10 from a 58 ng/µL stock.

  • V₁ = (C₂ × V_total)/C₁ = ((5.8 ng/µL) × 100 µL) / (58 ng/µL) = 10 µL stock; add 90 µL diluent.

• Example 2 (targeting 2 ng/µL from 58 ng/µL):

  • Overall factor = 2/58 ≈ 1/29. Create 1:10 (5.8 ng/µL) then 1:3 (≈1.93 ng/µL).
  • Step 1: 1:10 as above. Step 2 (from 5.8 ng/µL): V₁ = (2 ng/µL × 150 µL)/5.8 ng/µL ≈ 51.7 µL; add 98.3 µL diluent.

Quality Control & Acceptance Criteria

• Mixing: Each dilution should be homogeneous (vortex/pipette adequately).
• Replicates: Optional duplicates should agree within ±10–20% (context‑dependent).
• Contamination check: No detectable DNA signal in diluent blank for sensitive assays.
• Traceability: Record stock ID, initial concentration, diluent type, tube/plate map, operator, date/time.

Tips for Accuracy

• Use the largest practical volumes to minimize relative error.
• Keep DNA on ice; avoid repeated freeze–thaw.
• Pre‑wet tips and dispense below the meniscus, touching off to the tube wall.
• Change tips at every step; never pipette back into the source (avoid back‑contamination).
• For very low concentrations, prefer fluorometric quantification to confirm outcomes.

Waste Disposal

• Tips, tubes, and plates: dispose in appropriate laboratory waste per local policy.
• Liquids: collect and discard according to institutional chemical/biological waste procedures.