DNA Concentration Calculator

This DNA/RNA Concentration Calculator helps determine nucleic acid concentration and purity from spectrophotometer readings. It uses the Beer-Lambert law to calculate concentrations from absorbance measurements at 260nm and 280nm, provides purity ratios (260/280), and can calculate total yield. Supports dsDNA, ssDNA, and RNA calculations with appropriate extinction coefficients.

For optimal accuracy, keep A260 readings between 0.1 and 1.0. Use dilution if needed.

Nucleic Acid Type

A₂₆₀ Reading

A₂₈₀ Reading (optional)

Dilution Factor

Sample Volume (μL)

Tips:

Use pure water or buffer for dilutions
Ensure samples are well-mixed
Zero the spectrophotometer with your blank
For concentrated samples, use appropriate dilution

Results

Concentration:

25.00 μg/mL

0 μg/mLLow500+ μg/mL

Total Yield:

2.50 μg

DNA/RNA Concentration Calculator Documentation

Overview

Accurate determination of nucleic acid concentration is fundamental to molecular biology research, influencing the success of applications ranging from PCR and cloning to next-generation sequencing. This calculator uses spectrophotometric analysis, the most widely used approach for nucleic acid quantification, based on the principle that nucleic acids absorb ultraviolet light at specific wavelengths, primarily at 260 nm.

Scientific Principles

The quantification is based on the Beer-Lambert Law, which establishes a direct relationship between absorbance and concentration:

A = \varepsilon c l

Where:

A = Absorbance
ε = Molar extinction coefficient
c = Concentration
l = Path length (typically 1 cm)

Calculations

Basic Concentration

The concentration is calculated using the following formula:

\text{Concentration } (\mu g/mL) = A_{260} \times \text{Extinction Coefficient} \times \text{Dilution Factor}

Extinction coefficients for different nucleic acid types (at A₂₆₀ = 1.0):

Double-stranded DNA: 50 μg/mL (0.020 (μg/mL)⁻¹ cm⁻¹)
Single-stranded DNA: 33 μg/mL (0.027 (μg/mL)⁻¹ cm⁻¹)
RNA: 40 μg/mL (0.025 (μg/mL)⁻¹ cm⁻¹)

Molar Concentration

For applications requiring molar concentration:

\text{Molar concentration (nM)} = \frac{\text{ng/}\mu\text{L concentration} \times 10^6}{Y \times \text{length in bases}}

Where Y is the average molecular weight per nucleotide:

Double-stranded DNA: 660 g/mol
Single-stranded DNA: 330 g/mol
RNA: 345 g/mol

Purity Assessment

The purity ratio (A260/A280) indicates sample quality:

\text{Purity Ratio} = \frac{A_{260}}{A_{280}}

Expected purity ratio ranges:

Double-stranded DNA: 1.7 - 1.9
Single-stranded DNA: 1.7 - 1.9
RNA: 1.9 - 2.1

Individual nucleotides have different A260/A280 ratios:

Guanine: 1.15
Adenine: 4.50
Cytosine: 1.51
Thymine: 1.47
Uracil: 4.00

Limitations and Considerations

Spectrophotometric Limitations:

Cannot distinguish between DNA and RNA (both absorb at 260 nm)
Limited sensitivity for low-concentration samples (below 2-5 ng/μL)
Susceptible to interference from contaminants that absorb UV light
Reduced accuracy for short oligonucleotides

Common Contaminants:

Proteins (absorb at 280 nm, affecting A260/A280 ratio)
Phenol and organic solvents (absorb at 230 nm)
Guanidine salts from extraction procedures
Cross-contamination between DNA and RNA

Best Practices

Use pure water or buffer for dilutions
Ensure samples are well-mixed before measurement
Zero the spectrophotometer with your blank solution
For concentrated samples, use appropriate dilution to stay within the linear range (A260 between 0.1 and 1.0)
Consider using fluorescence-based methods for low-concentration samples
Purify samples before measurement to minimize contaminant interference
For critical applications, consider using multiple quantification methods