Quantification Reviewer (HY)

Nucleic Acid Quantification High Yield Reviewer

Core Concept

Nucleic acid quantification = measures concentration + purity of DNA/RNA after extraction, before proceeding to PCR.

Two Methods — Know the Difference

Spectrophotometry

Fluorometry

Measures

Light absorbed

Light emitted

Wavelength

260 nm

Varies by dye

Sensitivity

Moderate

High (detects lower concentrations)

Specificity

Low (contaminants interfere)

High (dye-specific)

Purity info

✅ Yes

❌ No

Cost

Cheap

Expensive

The Magic Number: 260 nm

  • DNA and RNA absorb UV light maximally at 260 nm

  • This is the basis of ALL spectrophotometric quantification

Nanodrop — Know Everything About This

Key facts:

  • UV spectrophotometer

  • Only needs 1–2 µL

  • No reagents needed

  • Range: 2 ng/µL to 15,000 ng/µL (no dilution needed)

Formula:

C = A₂₆₀ × 50 µg/mL × dilution factor

Constant to memorize:

A₂₆₀ of 1 = 50 µg/mL (for dsDNA)

Purity Ratios — MUST MEMORIZE

Sample

260/280

260/230

DNA

~1.8

~2.0

RNA

~2.0

~2.0

Contaminant cheat sheet:

  • Low 260/280 → Protein contamination (proteins absorb at 280 nm)

  • Low 260/230 → Salt or organic contamination (absorb at 230 nm)

  • Phenol contamination → absorbs at 270 nm

Ratios are pH dependent — measured in 10 mM Tris·Cl, pH 7.5

Diphenylamine (DPA) Method — 3 Key Facts

  1. DNA's 2-deoxyribose + diphenylamine + acid → blue colored complex

  2. Read at 595 nm

  3. Concentration determined by standard curve interpolation

  • Known concentrations plotted first → unknown samples calculated from curve

All Methods Compared

Method

Sensitivity

Key Advantage

Key Limitation

UV Spectrophotometry

2 ng/µL

Simple, fast, gives purity

Contaminants interfere

DPA Method

3 µg

Works with colorimeter/ELISA

Slow, laborious

Fluorometry

10–50 pg/µL

Most sensitive

No purity info, expensive

Electrophoresis

10 ng

Very specific (band size)

Slow, low accuracy

Absorption Wavelengths — Quick Reference

Substance

Wavelength

DNA / RNA

260 nm

Phenol

270 nm

Proteins

280 nm

Salts / Organics

230 nm

Fluorometry — Key Points Only

  • Dyes bind to nucleic acids → emit light when excited

  • Fluorescence intensity = proportional to nucleic acid amount

  • Different dyes for DNA, RNA, and proteins

  • More sensitive than spectrophotometry but cannot assess purity

Spectrophotometer Practical Tips (Likely Asked)

  • Clean pedestal before AND after use

  • Always measure the blank first

  • Re-blank every 30 minutes

  • Take duplicate measurements minimum

  • Watch out for air bubbles

One-Liner Rapid Fire

  • Quantification comes after extraction, before PCR

  • Nanodrop = fastest, smallest volume method

  • DPA = blue color, 595 nm, uses standard curve

  • Pure DNA 260/280 = 1.8 | Pure RNA 260/280 = 2.0

  • Both DNA and RNA 260/230 = 2.0

  • Fluorometry = most sensitive, least specific for purity

  • Low purity ratio = re-purify the sample