Understanding Spectrophotometry Using NanoDrop 2000: Measuring DNA Concentration and Purity

Introduction

Spectrophotometry is a widely used technique in molecular biology for measuring the concentration and purity of nucleic acids and proteins. The NanoDrop 2000, a compact microvolume spectrophotometer, allows these measurements using just 0.5–2 µL of sample — ideal for when sample availability is limited.

With the ability to scan from 190–840 nm, NanoDrop provides quick, accurate, and reliable absorbance data for DNA, RNA, proteins, dyes, and more, making it an indispensable tool in labs around the world.

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What Is Spectrophotometry and How Is It Used in This Setting?

Spectrophotometry is the measurement of how much light a substance absorbs at specific wavelengths. In biological labs, it’s used to:

• Quantify nucleic acids (DNA, RNA)
• Check sample purity
• Detect contaminants
• Assess labeling efficiency (for fluorescent probes)

The NanoDrop 2000 works by passing UV light through a tiny droplet of sample held between two optical fibers using surface tension. It detects the intensity of transmitted light and calculates absorbance, which is then translated into concentration using built-in formulas.

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What All Parameters Can Be Measured Using NanoDrop?

Parameter	Wavelength	What It Indicates
A260 (OD260)	260 nm	DNA/RNA concentration
A280 (OD280)	280 nm	Protein contamination
A230 (OD230)	230 nm	Salt/organic contaminant contamination
A340 (OD340)	340 nm	Background/baseline correction
260/280 Ratio	—	DNA vs. protein purity (normal range is between 1.8-2.0)
260/230 Ratio	—	DNA vs. chemical purity (ideal range is above 2.0)
Concentration	—	ng/µL, calculated from A260
Full spectrum	220–340 nm	Visual quality check

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Principles of Absorbance Photometry

The NanoDrop applies Beer–Lambert Law, which relates absorbance to concentration:

A = ε⋅c⋅l

Where:

• A = Absorbance (Optical Density)
• ε = Extinction coefficient (specific to each molecule)
• c = Concentration (mol/L or ng/µL)
• l = Pathlength (in cm; automatically adjusted in NanoDrop from 1.0 mm to 0.05 mm)

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What is Optical Density (OD)?

OD (Optical Density) or Absorbance is a logarithmic measurement of how much light is absorbed by a sample:

Where:

• I₀ = Incident light intensity
• I = Transmitted light intensity

OD values are used to quantify DNA and RNA by measuring absorbance at 260 nm.

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What Are the 260/280 and 260/230 Ratios? How to Interpret Them?

Ratio	Ideal Range	Interpretation
260/280	~1.8 (DNA), ~2.0 (RNA)	Lower values suggest protein contamination
260/230	~2.0–2.2	Lower values indicate chemical contaminants (e.g., salts, phenol, guanidine)
260/280 > 2.0	May occur with RNA contamination or very pure ssDNA; also if A280 is very low due to baseline fluctuation

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Why Are 260, 280, 230, and 340 nm Used?

Wavelength	What It Detects	Why Chosen?
260 nm	DNA & RNA	Nucleic acid bases (adenine, guanine, etc.) absorb maximally here
280 nm	Proteins	Aromatic amino acids like tryptophan/tyrosine absorb UV here
230 nm	Contaminants	Salts, phenol, EDTA, guanidine absorb strongly here
340 nm	Background	Used for baseline correction and scattering due to bubbles or dirty pedestal

In NanoDrop measurements, baseline correction is a method used to subtract background noise or absorbance that is not due to nucleic acids or proteins. The value at 340 nm is often used for this purpose because:

340 nm is a wavelength where nucleic acids and proteins do not absorb significantly.

Purpose of Baseline Correction at 340 nm:

• Corrects for background absorbance due to:
  • Dirty pedestal or cuvette
  • Bubbles
  • Residual solvents or buffer absorbance
  • Instrumental drift
• Improves the accuracy of the absorbance readings at 260 nm and 280 nm by subtracting any absorbance at 340 nm from the whole spectrum.

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How Is DNA Concentration Calculated Using OD?

NanoDrop uses a simplified Beer–Lambert equation with default extinction coefficients for nucleic acids:

Concentration (ng/µL)= A260 × ε

Molecule	Extinction Coefficient (ε)
dsDNA	50 ng·cm/µL
ssDNA	33 ng·cm/µL
RNA	40 ng·cm/µL

The NanoDrop auto-adjusts for path length and reports normalized results to a 10 mm path (1 cm).

1 absorbance unit at 260 nm (A260 = 1.0) corresponds to 50 ng/µL of dsDNA.

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Step-by-Step Guide: How to Use NanoDrop 2000

1. Setup

• Turn on the NanoDrop
• Open the software → Select “Nucleic Acid” → Choose sample type (DNA-50 for dsDNA)

2. Clean Pedestals

• Use a lint-free wipe and ethanol to clean both upper and lower pedestals

3. Blanking

• Load 1–2 µL of buffer (same buffer as used for the sample)
• Click Blank

4. Load Sample and Measure

• Pipette 1–2 µL of DNA onto the pedestal
• Lower the arm → Click Measure
• The software will show:
  • DNA concentration (ng/µL)
  • 260/280 and 260/230 ratios
  • Full spectrum from 220–340 nm

5. Clean After Use

• Wipe both pedestals thoroughly
• Repeat for the next sample

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Interpretation Table: What to Check After a Measurement

Parameter	Ideal Value	Indicates	Abnormality Suggests
A260	—	DNA present	Should be >0.1 for reliability
260/280	~1.8	Pure DNA	decreases in protein contamination; increases because of RNA or low A280
260/230	~2.0–2.2	No chemical contamination	↓ phenol/salt contamination
A340	~0	Background baseline	↑ suggests bubbles, smudges, or baseline drift
Full Spectrum	Peak at 260 nm	Expected for DNA	Shoulder or dip at 230/280 implies contamination

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Pro Tips

• If 260/280 > 2.0, suspect RNA carryover or extremely clean sample with low protein.
• If 260/230 < 1.5, check for leftover guanidine, phenol, or salts.
• Always use new aliquots for each measurement.
• Re-blank every 30 minutes for batch runs.

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