Effect of pH and ionic strength on the spectro-photometric assessment of nucleic acid purity
Effect of pH and ionic strength on the spectro-photometric assessment of nucleic acid purity
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DOI：
10.2144/97223st01
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发表时间：
1997-03-01
期刊：
影响因子：
2.7
通讯作者：
Chomczynski, P
中科院分区：
文献类型：
--
作者：
Wilfinger, WW;Mackey, K;Chomczynski, P
The ratio of absorbance at 260 and 280 nm (the A(260/280) ratio) isfreqnenrly used to assess the purity of RNA and DNA preparations. Data presented in this report demonstrate significant variability in the RNA A(260/280) ratio when different sources of water were used to perform the spectrophotometric determinations. Adjusting the pH of water used for spectrophotometric analysis from approximately 5.4 to a slightly alkaline pH of 7.5-8.5 significantly increased RNA A(260/280) ratio from approximately 1.5 to 2.0. Our studies revealed that changes in both the pH and ionic strength of the spectrophotometric solution influenced the A(260/280) ratios. In addition, the ability to detect protein contamination was significantly improved when RNA was spectrophotometrically analyzed in an alkaline solution. UV spectral scans showed that the 260-nm RNA absorbance maximum observed in water was shifted by 2 nm to a lower wavelength when determinations were carried out in Na2HPO4 buffer at a pH of 8.5. We found RNA A(260/280) ratios to be more reliable and reproducible when these spectrophotometric measurements were performed at pH 8.0-8.5 in 1-3 mM Na2HPO4 buffer.
