Biochemical Techniques and Instrumentation

Procedure

1. Take 1 ml TE buffer in a cuvette and calibrate the spectrophotometer at 260nm as well as 280nm.
2. Add 10μl of each DNA sample to 900μl TE (Tris-EDTA buffer) and mix well.
3. Use TE buffer as a blank in the other cuvette of the spectrophotometer.
4. Note the OD₂₆₀ and OD₂₈₀ values on spectrophotometer.
5. Calculate the OD₂₆₀/OD₂₈₀ ratio.
6. The amount of DNA can be quantified using the formula:

OD₂₆₀ x 100 (dilution factor) x 50 μg/ml

DNA concentration (μg/ml) =------------------------------------------------------

1000

Spectrophotomteric Conversions for Nucleic Acids:

1 A 260 of ds DNA = 50 μg/ml

1 A 260 of ss oligonucleotides = 33 μg/ml

1 A 260 of ss RNA = 40 μg/ml