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EFFECTS OF INSTRUMENT RESOLUTION ON ASSESSMENT OF DNA PURITY

ation of DNA purity. This communication addresses the effects of instrument bandwidth on spectrophotometric determination of DNA concentration and purity.


MATERIALS AND METHODS
Spectrophotometers used in this study were the Shimadzu models UV-2401PC (selectable bandwidth to 0.1nm), UV-1700 (fixed bandwidth, 1nm), and the UV-1240 (fixed bandwidth ,5nm). Calf thymus DNA and Bovine serum albumin were obtained from Sigma Chemical Company, St. Louis, MO. All other reagents were obtained from commercial sources and were of the highest available purity. Samples were prepared in 5mM KH2PO4 containing 145mM NaCl, pH 7.48. Semi-micro, far-UV quartz cells (~1.5mL) were used for all measurements.


RESULTS AND DISCUSSION
An example of the spectrophotometric determination of DNA is illustrated in Figure 1. In this figure, a sample of approximately 73 g/mL calf thymus DNA in physiological buffer was measured using a Shimadzu UV-2401 spectrophotometer with an instrument bandwidth of 1.0nm. As illustrated in Figure 1, this sample of pure DNA has an absorbance maximum (1.47) and minimum (0.62) at approximately 260 and 230nm, respectively. The width at half-height of this spectrum is approximately 43 nm.The spectrum of this same DNA sample was measured using instruments of 1 and 5nm fixed-instrument bandwidth. These data are shown in Figure 2.



The data in Figure 2 indicate that there is very little difference in the spectrum of pure, double-stranded DNA measured using spectrophotometers of 1 and 5nm instrument bandwidth. The absorbance of the sample at 260nm and the A260/A280 ratios are comparable and well within experimental error. A similar comparison of spectra of 1mg/mL Bovine serum albumin is shown in Figure 3.
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