Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity.
Abstrak
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.
Penulis (3)
W. Wilfinger
Karol Mackey
P. Chomczyński
Akses Cepat
- Tahun Terbit
- 1997
- Bahasa
- en
- Total Sitasi
- 539×
- Sumber Database
- Semantic Scholar
- DOI
- 10.2144/97223ST01
- Akses
- Open Access ✓