Effect of pH and Ionic Strength on the Spectrophotometric Assessment of Nucleic Acid Purity

Autor:	Karol Mackey, Piotr Chomczynski, William W. Wilfinger
Rok vydání:	1997
Předmět:	Measurement method Chromatography medicine.diagnostic_test RNA General Biochemistry Genetics and Molecular Biology Absorbance chemistry.chemical_compound chemistry Ionic strength RNA analysis Spectrophotometry Nucleic acid medicine DNA Biotechnology
Zdroj:	BioTechniques. 22:474-481
ISSN:	1940-9818 0736-6205
DOI:	10.2144/97223st01
Popis:	The ratio of absorbance at 260 and 280 nm (the A260/280 ratio) is frequently used to assess the purity of RNA and DNA preparations. Data presented in this report demonstrate significant variability in the RNA A260/280 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 A260/280 ratios 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 A260/280 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 A260/280 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.
Databáze:	OpenAIRE
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