Question

1. how do you determine DNA/RNA purity, how do you determine DNA concentration, how do you determine protein concentration, and how do you determine cell density?).

Answer 1

DNA concentration is estimated by measuring the absorbance at 260nm, adjusting the A260 measurement for turbidity (measured by absorbance at 320nm), multiplying by the dilution factor, and using the relationship that an A260 of 1.0 = 50µg/ml pure dsDNA.

Concentration (µg/ml) = (A260 reading – A320 reading) × dilution factor × 50µg/ml

DNA purity, measure absorbance from 230nm to 320nm to detect other possible contaminants. The most common purity calculation is the ratio of the absorbance at 260nm divided by the reading at 280nm. Good-quality DNA will have an A260/A280 ratio of 1.7–2.0. A reading of 1.6 does not render the DNA unsuitable for any application, but lower ratios indicate more contaminants are present. The ratio can be calculated after correcting for turbidity (absorbance at 320nm).

DNA purity (A260/A280) = (A260 reading – A320 reading) ÷ (A280 reading – A320 reading)

Three different methods for measuring protein concentration: absorbance at 280 nm, the Bradford assay, and the BCA assay.

Six methods to calculate cell densities: (1) a Neubauer improved hemacytometer, (2) an automated cell counter, (3) a manual-counting method, and three flow cytometry methods based on (4) autofluorescence, (5) propidium iodide staining, and (6) side scattered light (SSC).