Biotechnology and Bioinformatics (1+1)

B. Spectrophometric determination of DNA concentration

Dilute 1.5 μl of DNA to 1500 with deionised water and read at A₂₃₀, A₂₆₀ and A₂₈₀.

The A₂₆₀/A₂₈₀ ratio provides an estimate of the purity of the DNA. In a pure sample, this ratio is approximately 1.8. Lower values indicate protein or phenol contamination. A₂₃₀ should be less than A₂₆₀ and may be the same as A₂₈₀. High A₂₃₀ reading indicates that residual phenol remains in the preparation. An A₂₆₀ of 1 corresponds to approximately 50 μl/ml of double-stranded DNA in a 1 cm quartz cuvette. Nucleic acid concentration is calculated as follows:

A₂₆₀ * 50 mg/μl * 0.001 μl/ml * dilution factor (1500 μl/1.5 μl) (μg/μl)