OD260/OD280 Ratio Calculator

The OD260/OD280 Ratio Calculator measures the purity of DNA or RNA samples by comparing light absorbance at different wavelengths — a standard quality check in molecular biology labs before sequencing, cloning, or any downstream experiment. Select your ratio type (260/280 or 260/230), enter your absorbance readings (A260, A280, A230), and choose your nucleic acid type to get your Purity Ratio, sample Concentration, and a Purity Assessment indicating whether your sample is within acceptable limits.

Used for background correction (A260 - A320)

Cuvette path length in centimeters

Results

Purity Ratio

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Concentration

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Purity Assessment

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Corrected A260

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Frequently Asked Questions

What is a good OD260/OD280 ratio for DNA?

A pure DNA sample should have an OD260/OD280 ratio between 1.7-2.0. Ratios significantly lower than 1.7 may indicate protein contamination, while ratios higher than 2.0 may suggest RNA contamination or alkaline conditions.

What does the OD260/OD230 ratio indicate?

The OD260/OD230 ratio should be between 2.0-2.2 for pure nucleic acids. Lower ratios may indicate contamination with organic compounds like phenol, guanidine, or other chemicals that absorb at 230nm.

Why do we measure absorbance at 320nm?

A320 is used for background correction as nucleic acids don't absorb significantly at this wavelength. Subtracting A320 from other readings helps account for light scattering and turbidity in the sample.

How do I calculate nucleic acid concentration from OD260?

Concentration is calculated using the formula: (A260 × dilution factor × conversion factor) / path length. The conversion factor depends on the nucleic acid type: 50 μg/ml for dsDNA, 40 μg/ml for ssDNA/RNA, and 20 μg/ml for oligonucleotides.

What causes low OD260/OD280 ratios?

Low ratios (below 1.7) typically indicate protein contamination in your sample. Proteins absorb strongly at 280nm due to aromatic amino acids like tryptophan and tyrosine, increasing the denominator and lowering the ratio.

Should I use a dilution factor in my calculations?

Yes, if you diluted your sample before measurement, you must multiply by the dilution factor to get the actual concentration of your original sample. For example, if you diluted 1:10, use a dilution factor of 10.

What is the significance of different nucleic acid conversion factors?

Different nucleic acids have different extinction coefficients: dsDNA (50 μg/ml per OD unit), ssDNA and RNA (40 μg/ml per OD unit), and oligonucleotides (20 μg/ml per OD unit). Using the correct factor ensures accurate concentration calculations.