Safety

• Wear lab coat, gloves, and eye protection.
• Handle RNA with RNase‑free technique.
• Avoid skin contact with samples and cleaning solutions (e.g., 70% ethanol).
• Dispose of tips/wipes per institutional biosafety guidelines.

Materials and Equipment

• Micro‑volume spectrophotometer (e.g., NanoDrop or equivalent) with nucleic acid method.
• Computer with instrument software and printer (optional).
• Diluent/blank: Nuclease‑free water or low‑EDTA TE (match the sample buffer).
• Lint‑free laboratory wipes; 70% ethanol; DI/NUC‑free water for cleaning.
• Calibrated pipettes and aerosol‑resistant tips.
• Ice rack (optional, to stage samples).

Definitions & Key Formulae

• Baseline correction (optional): A*λ = Aλ − A320 (or A340, per instrument).

• Concentration (ng/µL):

  • dsDNA: Conc = A*260 × 50 × DF
  • RNA: Conc = A*260 × 40 × DF
  • ssDNA/oligos: Conc = A*260 × 33 × DF (or use sequence‑specific ε if known) DF = dilution factor used before measurement.

• Purity ratios (use baseline‑corrected values if applied):

  • DNA: A260/A280 ≈ 1.8 (acceptable ~1.7–1.9)
  • RNA: A260/A280 ≈ 2.0 (acceptable ~1.9–2.1)
  • All: A260/A230 ideally 2.0–2.2 Low A260/A280 suggests protein/phenol; low A260/A230 suggests salts, chaotropes, EDTA, carbohydrates.

Preparation

1. Warm‑up & self‑check: Power on per manufacturer guidance; run any required diagnostics if prompted.
2. Clean pedestals: Rinse with DI/NUC‑free water → wipe with lint‑free wipe → optional 70% ethanol → water → final wipe.
3. Verify blanks match sample buffer: Using water as blank for samples in TE can bias ratios; use the same buffer whenever possible.
4. Mix samples gently (pipette up/down or brief vortex) and quick spin to collect.

Procedure — Measurement

1. Launch software and select the nucleic acid method (choose sample type if prompted: dsDNA, RNA, etc.).

2. Baseline/blank:

   • Apply 1–2 µL of blank buffer to the lower pedestal.
   • Close arm and click Blank.
   • Open arm, wipe both pedestals clean.

3. Measure sample:

   • Apply 1–2 µL of sample. Ensure it fully bridges the pedestals; avoid bubbles.
   • Close arm and click Measure.
   • Record displayed A260, A280, A230 (and A320 if shown), concentration, ratios, and DF.
   • Open and wipe clean with a fresh lint‑free wipe.

4. Replicates: Measure each sample at least in duplicate (triplicate for critical work). Re‑pipette fresh aliquots for each read.

5. Carryover check: After high‑concentration or problematic samples, run a blank measurement to confirm the reading returns to near zero; re‑clean if not.

6. Dilution if needed: If software flags “over‑range”/non‑linear absorbance or concentration exceeds expected range, dilute the sample with matching buffer, record DF, and re‑measure.

Calculations & Reporting

• Use software‑reported concentration if baseline correction and sample type are correctly set. Otherwise:

  • A*260 = A260 − A320 (if A320 available; else use A260 directly).
  • Conc (ng/µL) = A*260 × factor × DF (factor = 50 dsDNA, 40 RNA, 33 ssDNA/approx).
  • Ratios: A260/A280 and A260/A230 using corrected absorbances if available.

• Example (dsDNA): A260=0.325, A280=0.180, A230=0.140, A320=0.005, DF=10 →

  • A*260=0.320; A*280=0.175; A*230=0.135 → Conc=0.320×50×10=160 ng/µL;
  • A260/A280=1.83; A260/A230=2.37 (acceptable).

Quality Control & Acceptance Criteria

• Blank check: Post‑cleaning blank absorbance should be ~0.000–0.005 A at 260 nm.

• Replicate agreement: Concentration replicates within ±5–10%.

• Purity thresholds (typical, adjust to assay needs):

  • DNA acceptable: A260/A280 ≥1.7; A260/A230 ≥1.8.
  • RNA acceptable: A260/A280 ≥1.9; A260/A230 ≥1.8.

• Trend review: Persistently low A260/A230 indicates residual salts/chaotropes—consider re‑purification.

• Controls (recommended): Measure a known‑concentration reference (DNA/RNA standard) weekly to verify accuracy.

Troubleshooting & Notes

• Low A260/A280 (<1.7 DNA / <1.9 RNA): Protein or phenol carryover. Re‑extract or clean up (e.g., additional wash/ethanol precip/column cleanup).
• Low A260/A230 (<1.8): Salts/guanidine/EDTA/carbohydrates. Additional wash or buffer exchange; ensure blank matches buffer.
• Ratios unusually high (>2.2): Very low ionic strength (e.g., water blank with DNA in water) or baseline drift; measure in TE or enable baseline correction.
• Unstable/erratic readings: Bubbles, insufficient volume, dirty pedestals, or sample not spanning pedestals. Re‑pipette, increase volume slightly, re‑clean.
• Very low concentrations (<~10 ng/µL): Absorbance becomes noise‑limited; confirm with a fluorometric assay (e.g., dye‑based).
• Very high concentrations: Dilute into the same buffer; record DF.
• RNA handling: Use RNase‑free tips/tubes; keep on ice; avoid repeated freeze–thaw.
• pH/ionic strength effects: Ratios shift with pH; TE (pH ~7.5–8.5) gives more stable readings than pure water.

Cleaning & Waste

• After each sample: wipe pedestals with a dry lint‑free wipe.
• Periodically: water → 70% ethanol → water; wipe dry.
• Dispose of tips and wipes in appropriate laboratory waste.

Quick Reference:

• dsDNA: Conc (ng/µL) = A*260 × 50 × DF; purity ~1.8 / >2.0 (A260/A280 / A260/A230).
• RNA: Conc (ng/µL) = A*260 × 40 × DF; purity ~2.0 / >2.0.
• Always blank with the sample’s buffer, enable A320 correction if available, and wipe between every sample.