Quantification Reviewer




Nucleic Acid Quantification Reviewer


What It Is & Why It Matters

Nucleic acid quantification measures the concentration and purity of DNA/RNA after extraction. It confirms you have enough quality genetic material before moving to downstream steps (PCR → gel electrophoresis). Without this step, experiments may fail or give inaccurate results.


Workflow in Mol Bio

Sample Collection → Nucleic Acid Extraction → Nucleic Acid Quantification → PCR Amplification → Gel Electrophoresis → Other Applications


Two Main Methods Spectrophotometry

  • Measures light absorbed by a sample at specific wavelengths

  • DNA/RNA absorb UV light strongly at 260 nm

  • Absorbance at 260 nm → used to calculate concentration

  • Fast, simple, no complex reagents needed

  • Moderate sensitivity; less specific (can be affected by contaminants)

Fluorometry

  • Uses fluorescent dyes that bind to nucleic acids and emit light when excited

  • Fluorescence intensity is proportional to nucleic acid amount

  • More sensitive and accurate at low concentrations

  • Different dyes are selective for DNA, RNA, or proteins

  • More expensive; requires fluorescent labeling; no purity info


Spectrophotometry vs. Fluorometry (Quick Compare)

Feature

Spectrophotometry

Fluorometry

Principle

Absorbance of light

Emission of fluorescence

Sensitivity

Moderate (~2 ng/µL)

High (10–50 pg/µL)

Specificity

Low (affected by contaminants)

High (nucleic acid-specific)

Purity info

Yes

No

Cost

Lower

Higher


Spectrophotometric Methods

1. Nanodrop Method

  • Type: UV spectrophotometer

  • Sample volume: 1–2 µL (microvolumes)

  • No additional reagents needed; quick and easy

  • Principle: Light absorbed at 260 nm is proportional to nucleic acid present

  • Constant for dsDNA: A₂₆₀ of 1 = 50 µg/mL

  • Formula: C = A₂₆₀ × 50 µg/mL × dilution factor

  • Concentration range: 2 ng/µL – 15,000 ng/µL (no dilution needed)


What it measures:


  • Concentration (ng/µL)

  • Protein contamination → 260/280 ratio

  • Salt contamination → 260/230 ratio


2. Diphenylamine (DPA) Method

  • The 2-deoxyribose of DNA reacts with diphenylamine in acid → forms a blue colored complex

  • Read at 595 nm using a spectrophotometer

  • Concentration calculated by interpolating from a standard curve

  • Known DNA concentrations are measured first → absorbance values plotted → unknown samples calculated from the curve

  • Sensitivity: ~3 µg

  • Can be measured with a colorimeter or ELISA reader

  • Limitation: Laborious and time-consuming


DNA Purity — Critical Ratios (MEMORIZE)

Sample

260/230

260/280

DNA

2.00 (2.00–2.20)

1.80 (1.80–2.00)

RNA

2.00 (2.00–2.20)

2.00 (1.80–2.00)


Contaminant indicators:


  • A230 → Organic substances

  • A270 → Phenol

  • A280 → Proteins


Note: Ratios are pH dependent — use 10 mM Tris·Cl, pH 7.5. Deviations from optimal ratios signal contamination and possible need for re-purification.


Comparison of All DNA Quantification Methods

Method

Sensitivity

Advantages

Limitations

UV Spectrophotometry

2 ng/µL

Simple, fast

Low specificity, sensitive to contaminants

Diphenylamine (DPA)

3 µg

Usable with colorimeter/ELISA

Laborious, time-consuming

Fluorescence

10–50 pg/µL

High specificity

Expensive reagents

Electrophoresis

10 ng

Very high specificity (band size)

Time-consuming, low accuracy


Spectrophotometer Tips (Practical)

  1. Always clean the pedestal before use

  2. Always measure your blank first

  3. Check for air bubbles

  4. Mix samples before measuring

  5. Be careful with low concentration samples

  6. Re-blank every 30 minutes

  7. Take measurements in at least duplicates

  8. Always write down your results

  9. Clean the pedestal after use


High-Yield Points for the Exam

  • DNA/RNA absorb maximally at 260 nm

  • Proteins absorb at 280 nm; phenol at 270 nm; salts/organics at 230 nm

  • Pure DNA = 260/280 ratio of ~1.8

  • Pure RNA = 260/280 ratio of ~2.0

  • Both DNA and RNA have optimal 260/230 ratio of ~2.0

  • Nanodrop uses only 1–2 µL with no reagents

  • DPA method produces a blue complex read at 595 nm

  • Fluorometry is more sensitive but gives no purity information