Calculate the concentration and total yield of plasmid DNA F

Calculate the concentration and total yield of plasmid DNA

Fraction of Elution

Absorbance at 280.4 nm

Absorbance at 489.4 nm

1

0.029

-0.009

2

0.555

0.115

3

0.488

-0.008

4

0.482

-0.012

5

0.486

-0.008

Solution

Answer: The concentration of the DNA and total yield cannot be calculated for the above given readings at Abs 280.4 nm and Abs 489.4 nm.

The Concentration of any DNA (either plasmid or any other genomic DNA) is estimated by measuring the absorbance in spectrophotometer at 260nm and by multiplying with the dilution factor.

Inorder to evaluate the purity of the DNA, the absorbance should be from 230nm to 320nm. 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. Lower ratios indicates that 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)

Total yield is obtained by multiplying the DNA concentration by the final total purified sample volume.

DNA yield (µg) = DNA concentration × total sample volume (ml)