Determination of Concentration of Purified DNA:
After purification the integrity of purified sample can be checked on agarose gel. The concentration (μg cm -3 ) of DNA can be estimated by spectrophotometer.
50 XA260 = Concentration of DNA Sample
| Type of Nucleic Acid |
Extinction Coefficient [cm -1 M -1 ] |
| Double Stranded DNA |
50 |
| Single Stranded DNA |
33 |
RNA |
40 |
DNA is the polymer of nucleotides consists of pentose sugar, nitrogenous bases (A, T, G, C) and phosphate. The nitrogenous bases contain aromatic ring structure, which absorbs maximum at 260 nm, this counts the uv-spectrophotometric DNA estimation. The possible contaminants of DNA (protein & lipids) and reagents used during DNA preparation (phenols) also absorbs at 280 nm. The purity of DNA sample can be determined by the ratio of A260 to A280 . If the ratio is ≥ 1.8, means DNA is comparatively in pure form.
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