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EFFECTS OF INSTRUMENT RESOLUTION ON ASSESSMENT OF DNA PURITY

his technique.

A series of samples with varying ratios of DNA and BSA were prepared in physiological buffer, and A260/A280 ratios for these samples were measured in spectrophotometers with 1 and 5nm bandwidth. The results are presented in Table 1.


The 1nm bandpass instrument was capable of discriminating a decrease in the A260/A280 ratio of DNA samples containing 10% by weight BSA, or contamination on the order of 10 g. This capability is more readily appreciated by examination of the data in Figure 5.


There appears to be little discernable change in the A260/A280 ratio of DNA-Protein mixtures measured using the 5nm bandpass instrument until approximately 20% by weight BSA or approximately twice the minimum level detected by the 1nm bandpass instrument.


SUMMARY
Both 1nm and 5nm bandpass spectrophotometers are appropriate for measurement of pure DNA samples. But for determination of DNA purity, particularly at very low (~10g) amounts of protein, there are significant advantages to the use of a 1nm bandpass spectrophotometer.


REFERENCES
1.Glasel, J.A., Validity of Nucleic Acid Purities Monitored by A260/A280 Absorbance Ratios, Biotechniques 18:62-63, 1995.

2. Maniatis T., E.F. Fritsch, and J. Sambrook, Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Springs Harbor, NY, 1982



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