DNA and RNA quantitation is performed using various methods.
- Absorbance/spectrophotometric methods
- Gel electrophoresis – Agarose gel electrophoresis
- Fluorometric methods
Absorbance method (UV spectrophotometer, NanoDrop)
- At 260 nm nucleic acids absorbs light most strongly
- At 280 nm proteins are absorbs light most strongly
- At 230 nm other chemical contamination absorbs light most strongly.
DNA purity (protein contaminants) = A260 /A280
- High quality DNA will have an A260/A280 ratio of 1.7–2.0 (≈ 1.8)
- High quality RNA will have an A260/A280 ratio of ≈ 2.0
DNA purity (chemical contaminants) = A260 /A230
- Expected 260/230 values in the range of 2.0–2.2
- The reasons for the low A260 /A230 ratio is residuals of phenol, carbohydrate, guanidine.
Concentration (μg/mL) = (A260 measurement) x nucleic acid conversion factor x dilution factor
- DNA yield (μg) = DNA concentration × total sample volume (mL)
Fluorometric methods (Qubit fluorometers)
- Fluorescent dyes intercalate and bind nucleic acid grooves,
- Fluorometric instrument detect fluorescent signals of bound dyes
and measure nucleic acid concentration.
Gel Electrophoresis methods
Most common and widely used method.
Good quality genomic DNA should migrate as a high molecular weighted bands with little or no smearing.
Good quality RNA have 2:1 (28S/25S:18S) ratio.
