During several different stages of molecular cloning, it is important to get a quick and accurate reading of DNA concentration and purity. This can be done with a spectrophotometer. A spectrophotometer uses the absorbance/transmission of light through a liquid to determine the concentration of a particular substance in that liquid. Molecules absorb different wavelengths of light to varying degrees and most have a specific wavelength that they maximally absorb. By measuring the absorbance of a liquid you can accurately estimate the concentration of a substance in that liquid.
In order to accurately measure the concentration of a substance based on its absorbance, you need to know the wavelength of light that your substance maximally absorbs. In the case of nucleic acid (DNA and RNA), the maximal absorbance is at 260nm. Protein maximally absorbs at 280nm and the ratio of nucleic acid to protein (260/280) is generally used as an indicator of the purity of DNA samples.
These days, many labs have a NanoDrop, which is a very small spectrophotometer that can accurately read DNA concentration and purity in as little as 1μl. Regardless of whether you have a NanoDrop, follow the manufacturer's instructions for the spectrophotometer specific to your lab.
- Before measuring any samples, be sure to ‘blank’ the spectrophotometer using the solution the DNA is resuspended in, but with no DNA added. 'Blanking' measures the background inherent to the machine and your solvent.
- If using a NanoDrop to measure your samples, place 1-2µL of mini-prepped DNA onto the pedestal.
Close the lid and click measure, be sure to record the concentration and purity.
Repeat for each sample.
Note: Purity is measured under the 260/280 column (A good purity ranges from 1.80-2.00).
Note: Keep in mind that despite the accuracy of the NanoDrop, if two consecutive samples have significantly different concentrations, it is possible that the difference between them has affected the accuracy of the NanoDrop. It is a good idea to re-zero any spectrophotometer between samples if they are expected to vary significantly in concentration.
Note: DNA dissolved in water is going to have a greater variance in concentration readings than a DNA sample dissolved in buffer (such as TE). You will get much more accurate and consistent readings from DNA in a buffered solution.