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EFFECTS OF INSTRUMENT RESOLUTION ON ASSESSMENT OF DNA PURITY


The spectra in Figure 3 have been offset by 0.1A in order to show the enhanced details in the spectrum of serum albumin measured in the 1nm bandwidth instrument. With enhanced resolution, the contributions from phenylalanine, tyrosine, and tryptophan become more obvious. These residues absorb between 260 and 280nm. However, as was the case for the sample of pure DNA, there was little difference in the measured absorbance (~0.6) and wavelength maximum (~278nm) of the protein sample in the two instruments.

A much different situation applies when DNA purity is assessed. In this situation, the instrument must discriminate between DNA and likely contaminants. These contaminants include protein, chloroform, phenol. Contamination by protein is usually indicated by a decrease in the A260/A280 ratio. Contamination by an extraction solvent such as phenol or chloroform is indicated by increased absorbance at 320nm. If the criterion for acceptable resolution of instrument bandwidth ten-fold less than peak wavelength difference (280-260nm = 20nm) is applied to samples containing DNA and protein, then an instrument with a bandwidth of = 2nm should be used. In this case, there are demonstrative advantages in using a spectrophotometer with a bandwidth of 1nm. The spectra of a sample containing 36g/mL DNA and 500 g/mL serum albumin are shown in Figure 4.


It is clear from these data that the A260/A280 ratios of the sample containing both DNA and protein measured in the 5nm bandpass instrument (1.11) and in the 1nm bandpass instrument (1.02) are not equivalent. While both ratios are significantly less than the value of 1.8 expected for pure DNA, this was a sample heavily weighted with protein (~93% by weight). A more interesting question is the lowest level of protein in a sample that can be determined by t
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