How pure is your DNA?

So once you have isolated genomic DNA from some peas, you need a way to assess how much DNA you have got, and how pure you have managed to get it....

This can be done using a spectrophotometer taking advantage of the different absorbance spectrums of DNA and the common contaminants, RNA and proteins.

As I have shown in the diagram of an absorbance spectrum below, DNA absorbs light most at a wavelength of 260nm, protein absorbs light most at 280nm. A DNA/Protein mixture on the other hand will have a spectrum somewhere in-between - exactly where this spectrum will be depend on how much DNA vs protein there is in the sample.

So when we are assessing how much DNA we have, the absorbance of the sample at 260nm can be used to estimate this. 

And when we are assessing the purity of the sample (i.e. how much protein contamination we have), we can find the ratio of the absorbance of the sample at 260nm : the absorbance at 280nm (in other words you divide the absorbance at 260nm by the absorbance at 280nm).....

If you get...

• A260/A280 = 1.8   This means your DNA is pure
• A260/280 = 0.5  This means your sample contains mainly protein
• A260/A280 = between 0.5 - 1.8  This means your DNA is contaminated with protein - the nearer the value is to 0.5, the more protein contamination you have
• A260/280 = greater than 1.8  This means your sample is contaminated with RNA (because the uracil bases in RNA absorb more 260nm light than the thymine bases they have replaced)

:-)