The best way to determine the purity of my DNA?
The ratio of absorbance at 260 nm to the absorbance at 280 nm (A260/A280) is typically used to measure the purity of the sample. This is because DNA absorbs light most strongly at 260nm so the absorbance value at this wavelength can be used to estimate the DNA concentration using an equation derived from “Beer’s Law”. A ratio of ~1.8 is generally accepted as pure for DNA, whereas a ratio of ~2.0 is generally accepted as pure for RNA, however as the aromatic amino acids, tyrosine and tryptophan absorb strongly at 280 nm, a decrease in the ratio is used as an indicator of protein contamination.
The A260/A230 is another indicator of contaminants, the expected values are commonly in the range of 2.0-2.2, Contaminants such as EDTA, carbohydrates, phenols and have an absorbance close to 230 nm and so will decrease the ratio, whereas guanidine isothiocyanate will increase the ratio. A poorer ratio is therefore often found when extracting using nucleic acid purification columns, although this may not interfere with downstream processes.