Spectrophotometry

used to determine: the quantity and quality of nucleic acids (DNA and RNA)

Principle:

• every substance has a maximum absorbance to a certain wavelength of light

  • absorbed light is converted into energy

  • unabsorbed light is reflected away from surface

• a spectrophotometer measures the amount of light that a sample absorbs

• the instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector on the other end

• higher absorbance = higher optical density = more concentrated

Note: The light should penetrate through the clear sides of cuvettes

• use a blank to calibrate the spectrophotometer to zero OD

  • cuvette and solvent used to dissolve the protein samples may absorb light of the same wavelength as sample → false high reading

  • actual OD sample = OD sample + solvent - OD solvent

• measurement nucleic acid concentration

  • DNA and RNA absorb light of 260 in wavelength

  • each 50 microlit per ml of DNA gives an OD reading of 1

  • each 40 microlit per ml of RNA gives an OD reading of 1

Ratio of OD 260 / OD 280 - to determine DNA purity

• proteins absorb light of 280nm in wavelength

• high OD280 reading = high protein content

• OD 260/280 = 1.8 - 2.0 indicates good DNA purity

• Ratio < 1.8 indicates protein contamination

• Ratio >2.0 indicates RNA contamination