WHAT IS THE A260/A280 RATIO?
This ratio is used to determine the purity of a DNA or protein sample. Contamination of a nucleic acid solution by proteins, carbohydrates, and other organic molecules can be determined using a procedure called the A260/A280 ratio.
The basis of this test relies on the Beer-Lambert Law: A = ebc; where A is absorbance, e is the molar extinction coefficient, b is the cell path length, and c is the sample concentration. The commonly accepted average extinction coefficients for a 1 mg/mL nucleic acid solution at 260 nm and 280 nm are 20 and 10, respectively. In proteins, the extinction coefficient values at 260 nm and 280 nm at a concentration of 1 mg/mL are 0.57 and 1.00, respectively. Therefore, nucleic acid samples would be expected to have a higher absorbance at 260 nm than at 280 nm; in a protein sample, the opposite is true. Using these extinction coefficients, pure nucleic acid samples would have an A260/A280 ratio of 2.0, while protein would be 0.57.
HOW DO I GET A260/A280 USING LOGGER PRO?
1) Make sure you are using a UV-VIS spectrophotometer (such as the Vernier UV-VIS Spectrophotometer (VSP-UV)) with quartz cuvettes. Your sample should not be turbid or have any particulates in it. Please be familiar with operation of the device beforehand by reading the user manual (Specifications and User Guide).
2) Make sure a proper background measurement has been made using a solution without the DNA and/or protein of interest. Examples are water and buffer.
3) Collect an absorbance spectrum of your sample. Make sure the maximum absorbance value is between 0.1 and 1.0 absorbance units. Any values outside this range may result in decreased accuracy.
4) Using the examine feature, or by scrolling down the data table, find the absorbance values at 260 nm, 280 nm, and 340 nm.
5) Subtract the 260 nm and 280 nm absorbance values by the value at 340 nm. You can pick a different absorbance value to normalize by other than 340 nm; this is simply the typical value used in reference protocols.
6) Divide the resulting 260 nm value by the 280 nm value. If you are concerned with your resulting value, see the explanation above for what to expect.
You can also have Logger Pro report the absorbance readings at 260, 280, and 340 nm without collecting a full spectrum each time. Place your sample in the spectrometer. Go the Spectrometer Dialog and change the Collection Mode to Absorbance vs Concentration. Change “Single 10 nm Band” to Individual Wavelengths and select the wavelengths that are closest to 260, 280, and 340 nm. Select OK and then begin data collection. The absorbance values at 260, 280, and 340 nm will be recorded in the table.
**A free experimental write-up for this type of experiment is available on the Vernier UV-VIS Spectrophotometer’s main webpage: Vernier UV-VIS Spectrophotometer (VSP-UV)