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1. Quantification of nucleic acids
1 OD260 of dsDNA in water equal to 50 µg/ml; pure
DNA has an A260/A280 ratio of 1.8-2.0 in 10 mM
Tris pH 8.5.
1 OD260 of ssDNA in water equal to 33 µg/ml;
1 OD260 of ssDNA oligonucleotide in water equal to 20-30
µg/ml;
1 OD260 of ssRNA in water equal to 40 µg/ml; pure
RNA has an A260/A280 ratio of 1.9-2.1 in 10 mM
Tris pH 7.5.
OD value should be between 0,1 and 1,0 to ensure an optimal measurement.
Absorbance at 230 nm and 270 nm indicates the presence of phenol or
urea; absorbance at 280 nm (and as consequence of this, A260/A280
<1.8) indicates the presence of protein; A260/A280
>2 indicates a possible contamination with RNA; absorbance at 325
nm indicates the presence of particulates or duty cuvettes.
2. Quantification of nucleotides
max. absorbance at indicated wavelength and pH 7.0:
dGTP 253 nm
dATP 259 nm
dTTP 267 nm
dCTP 271 nm
Conversion formula:
ODmax
c = —————,
Eco
where c is the concentration
in nM; ODmax is observed absorbance
at above indicated wavelength; Eco is the
extinction coefficient for certain nucleotide.
3. Quantification of proteins
- 1 OD280 of proteins in water equal to 1 mg/ml (for ranges
of protein concentrations from 20 to 3000 µg/ml); protein concentration
= has an A205/31 (for ranges of protein concentrations
from 1 to 100 µg/ml).
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