Quantification Reviewer (HY)

HIGH-YIELD EXAM REVIEWER Nucleic Acid Quantification | MLS 420

MUST-KNOW CONCEPTS The Golden Rules

  1. Impure sample = Repeat entire extraction (no exceptions, even if concentration is good)

  2. Bad quality DNA on gel = Repeat entire extraction

  3. Both methods give concentration, ONLY spectrophotometry gives purity ratios

  4. Fluorometry is 1,000–10,000× more sensitive than spectrophotometry

SPECTROPHOTOMETRY vs FLUOROMETRY

(Most likely to be tested as MCQ)

Spectrophotometry

Fluorometry

Measures

Absorbance

Emitted light intensity

Principle

Beer-Lambert's Law

Fluorophore-target binding

Sensitivity

Moderate

HIGH

Specificity

Low (contaminants interfere)

HIGH (target-specific)

Purity ratios

YES

NO

Cost

Cheap

Expensive

Prep time

Fast

Slow/tedious

Availability

Common in PH

Less common

NANODROP — HIGH YIELD FACTS

Formula (memorize this):

C = A₂₆₀ × 50 µg/mL × Dilution Factor

  • Wavelength for nucleic acids = 260 nm

  • Wavelength for proteins = 280 nm

  • Wavelength for salts/organics = 230 nm

  • Wavelength for phenol = 270 nm

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

  • Volume needed: 1–2 µL only

  • No reagents needed

Sample calculation:

  • A₂₆₀ = 0.5, DF = 10

  • C = 0.5 × 50 × 10 = 250 µg/mL

PURITY RATIOS — MEMORIZE THESE

Ratio

Detects

DNA Optimal

RNA Optimal

260/280

Protein contamination

~1.8

~2.0

260/230

Salt/organic contamination

~2.0

~2.0

Trick to remember:

  • 260/280 → 8 comes before 0 → DNA = 1.8, RNA = 2.0

  • 260/230 → Both DNA and RNA = 2.0

  • Any value below OR above the range = IMPURE

GEL ELECTROPHORESIS INTERPRETATION

Observation

Meaning

Action

Smeared/smudged band

Degraded DNA

Repeat extraction

No bands

Low/insufficient concentration

Concentrate → if still low, repeat

DNA won't migrate (stays in well)

Excess/too high concentration

Dilute

Intact, clear band

Good quality DNA

Proceed

CONCENTRATION INTERPRETATION FOR PCR

Concentration

Meaning

Action

< 5 ng/mL

Too low for PCR

Concentrate first → repeat if still low

5–50 ng/mL

IDEAL for PCR

Proceed

> 50 ng/mL

Too high

Dilute

PRACTICE TEST — RAPID INTERPRETATION

Know this pattern cold:

Value

Status

Reason

260/280 = 1.98

Pure

Within 1.8–2.0 range

260/280 = 1.72

Impure

Below 1.8 → protein contamination

260/280 = 1.32

Impure

Far below 1.8 → protein contamination

260/230 = 2.10

Pure

Within 2.0–2.20 range

260/230 = 0.21

Impure

Far below 2.0 → salt contamination

260/230 = 0.71

Impure

Below 2.0 → salt contamination

Conc = 258.83 ng/µL

Too HIGH

Dilute before PCR

Conc = 53.78 ng/µL

Slightly high + impure

Repeat extraction

Conc = 22.43 ng/µL

Good range but impure

Repeat extraction

Bottom line: Impurity ALWAYS overrides concentration. If impure → repeat, period.

DIPHENYLAMINE METHOD — ONE LINER

2-deoxyribose + acid → reacts with diphenylamine → blue complex → read at 595 nm → blue intensity ∝ DNA concentration → plot on standard curve

BEER-LAMBERT'S LAW

A = log₁₀ (I₀/I) = εcl

  • Absorbance ∝ Concentration (DIRECT)

  • Transmitted light ∝ Concentration (INVERSE)

  • High concentration → more light absorbed → less light transmitted

FLUOROMETRY PRINCIPLE — ONE LINER

Fluorophore binds specifically to target → excitation → emits light → intensity of emitted light ∝ DNA concentration

  • High intensity = High concentration

  • Low/faded light = Low concentration

  • No binding = No fluorescence

RAPID-FIRE FACTS

  • Nanodrop uses UV spectrophotometry

  • Standard drop = 20–25 µL | Nanodrop = 1–2 µL

  • Correct drop shape on pedestal = perfect dome (Figure B)

  • Re-blank spectrophotometer every 30 minutes

  • Always measure at least in duplicates

  • PCR is the most common downstream application requiring exact concentration

  • DNA isolation = most crucial step in molecular biology

  • Phenol contaminant detected at 270 nm (between 260 and 280 — easy to miss)

  • Fluorometry needs fluorescence labeling → longer prep time = major disadvantage

  • Spectrophotometry can analyze wide range of compounds, fluorometry cannot

TOP EXAM TRAPS

❗ Trap 1: Sample is pure but concentration is too high → dilute, don't repeat

❗ Trap 2: Sample has good concentration but is impure → repeat extraction, don't just dilute

❗ Trap 3: Fluorometry is more sensitive but cannot give purity ratios — examiners love this

❗ Trap 4: 260/280 optimal for DNA is 1.8, not 2.0 (2.0 is for RNA)

❗ Trap 5: Diphenylamine is read at 595 nm, NOT 260 nm

❗ Trap 6: Dilution factor in Nanodrop formula is optional — only applied when sample was diluted

❗ Trap 7: Both methods give concentration — the difference is purity ratios, NOT concentration