Calculate DNA and RNA concentration from absorbance at 260nm
DNA concentration is calculated using the Beer-Lambert Law: A = εlc, where A is absorbance at 260 nm, ε is the extinction coefficient, l is the path length, and c is concentration. For double-stranded DNA, an A260 of 1.0 equals 50 µg/mL. The calculator applies the appropriate coefficient based on your nucleic acid type.
Accurate DNA concentration measurement is critical for downstream applications like PCR, cloning, sequencing, and transfection. Too little DNA leads to failed experiments; too much can inhibit enzymatic reactions. Precise quantification ensures reproducible results, saves expensive reagents, and prevents weeks of troubleshooting failed experiments in molecular biology laboratories.
The A260/A280 ratio assesses nucleic acid purity. Pure DNA has a ratio of ~1.8; pure RNA is ~2.0. Ratios below these indicate protein contamination (proteins absorb at 280 nm). The A260/A230 ratio checks for organic solvent contamination—values of 2.0-2.2 indicate clean samples. Both ratios together give a complete picture of sample quality.
Blank your spectrophotometer with the same buffer used to dissolve your DNA. Ensure absorbance readings fall between 0.1 and 1.0 for accuracy—dilute concentrated samples. Use nuclease-free water and clean cuvettes. For very low concentrations, fluorometric methods (like Qubit) are more accurate than spectrophotometry. Always measure in duplicate for reliability.
Spectrophotometric measurement at 260 nm (A260) is the standard method for quantifying nucleic acids. Nucleic acids have an absorption maximum at 260 nm due to the aromatic rings in their nitrogenous bases.
Indicates protein contamination:
Indicates organic compound contamination:
The A260/A280 ratio measures nucleic acid purity by comparing absorbance at 260 nm (where DNA absorbs) to 280 nm (where proteins absorb). Pure DNA has a ratio of approximately 1.8, and pure RNA has a ratio of approximately 2.0. A ratio below 1.6 indicates significant protein contamination, while a ratio above 2.0 in a DNA sample may indicate RNA contamination. This ratio is the most common quick check for sample quality.
For standard PCR, 1 to 10 nanograms of genomic DNA template per 50-microliter reaction is typical. For plasmid DNA, 0.1 to 1 nanogram is usually sufficient. Too much template DNA can inhibit the reaction or produce non-specific amplification, while too little may result in no product. Always check your specific protocol, as optimal concentrations vary depending on the polymerase, target sequence, and application.
Use the dilution formula C1V1 = C2V2, where C1 is your current concentration, V1 is the volume of DNA to use, C2 is your desired concentration, and V2 is the final volume. For example, to dilute 100 ng/uL DNA to 10 ng/uL in a final volume of 50 uL, calculate V1 = (10 x 50) / 100 = 5 uL of DNA stock, then add 45 uL of nuclease-free water or TE buffer.