- DNA sample
- SSC buffer
- UV spectrophotometer and quartz cuvettes
- Dissolve a small quantity of your extracted DNA in 3.0 mL of 0.1X SSC.
- Turn on and blank a UV spectrophotometer at 220 nm (use 0.1X SSC as
the blank). Determine the absorbance of your sample DNA at 230 nm.
- Change the wavelength to 230 nm, reblank the spectrophotometer, and
measure the absorbance of the sample at 230 nm.
- Increment the wavelength by 10 nm and repeat blanking and measuring
the absorbance until readings are taken through 300 nm.
- Compute the absorbance ratio 260 nm to 280 nm. Pure DNA (without protein
or RNA) will have a 260:280 absorbance ratio of 1.85. RNA will have a
260:280 ratio of 2.0.
- Plot the absorbance spectrum of your sample and indicate the 260:280
ratio, as well as the amount of protein contamination on the graph.