Question

OD o X Load your sample and press the measure button. Blank Re-Blank Sample ID: Pedestal ort pectra ple volume Type: DNA 50.00 Conc. 23.7 ng/ul - 10 mm Off 37°C A260 (10 mm path) 0.473 A280 (10 mm path) 10.320 260/2801 .48 260/230 0.32 1 Baseline correction 340 nm 10mm Absorbance 001 220 230 240 250 260 270 280 290 Wavelength (nm) 300 310 320 330 340 350 285nm 0.274Abs sure Nucleic Acid

After purification of your favorite plasmid you use a spectrophotometer to find the concentration of your DNA and observe the graph below. Resketch by hand this graph on your answer page with exact axis labels and scales. Redraw the red line. Draw, in your estimation, the absorption spectrum of what is expected of a good DNA prep.

Answer 1

Spectrophotometric analysis is a commonly used method to quantify nucleic acids (DNA and RNA). Nucleic acids show maximum absorption of light in the ultraviolet spectrum, at 260 nm. In a spectrophotometric graph, the wavelength is plotted on X-axis and the absorption is plotted on the Y-axis.

Since the nucleic acids show maximum absorption at 260 nm, the peak of the spectrophotometric graph is generally around this wavelength. In the graph given in the question, the trend of the absorption spectrum of plasmid DNA doesn't show a peak, possibly due to the presence of contaminants and substantially lower amounts of DNA. A normally expected trend of the absorption spectrum of a good DNA prep is shown in black in the below hand-drawn graph.

1.2 2.0 18-1 1.6 10m Absorbance 1.07 0.87 0. 61 0.4. 0.2- 0.0.1 220 230 240 250 260 270 280 290 300 310 320 330 340 350 Warelength (mm)

The image given in the question also shows the values of 260/280 and 260/230 ratios which are the ratios of the absorbance of the sample at those particular wavelengths (i.e. 230, 260 and 280 nm). These ratios are generally considered for assessing the purity of the given nucleic acid sample. Values of 1.8 and 2.0-2.2 for the 260/280 and 260/230 ratios, respectively, are generally considered as standards for 'pure' DNA samples. It is worth noting that the 260/280 and 260/230 values in the question are lower than these standards, possibly due to the presence of contaminants.