Use Of A Spectrophotometer To Quantify And Determine Purity
Of Nucleic Acids
Common cellular components such as nucleic acids and proteins absorb light in the
ultraviolet (UV) range.
This fact can be used to quantify and determine the purity of nucleic acids isolated
from cells.
Contaminants such as proteins and phenols will absorb light at a wavelength of 230
nm.
Nucleic acids will absorb light at a wavelength of 260 nm.
Proteins will absorb light at a wavelength of 280 nm.
Particulate matter and dirt will absorb light at a wavelength of 325 nm.
The A260/A280 ratio can be used to determine the purity of a nucleic acid sample.
Highly purified DNA samples will typically have A260/A280 ratios ranging from 1.8 to 1.9
Highly purified RNA samples will typically have A260/A280 ratios ranging from 1.9 to 2.0
A260 value of a nucleic acid sample can be used to determine its concentration.
An A260 value of 1.0 indicates the following concentrations:
[dsDNA] = 50mg/ml
[ssDNA] = 37mg/ml
[ssRNA] = 40mg/ml