Molecular Research Center Phosphate Buffers for RNA spectrophotometry Phosphate Buffer

PHOSPHATE BUFFER
Cat. No. SP 130
Store at room temperature. Phosphate Buffer is stable at least 18 months after date of purchase.

PRODUCT DESCRIPTION
Phosphate Buffer is a stock solution containing 0.3 M Na2HPO4 for use in adjusting distilled, deionized or filtered water to a pH of 8.4 - 8.6. This slightly alkaline pH of water used to dilute RNA and DNA samples for spectrophotometric analysis reduces spurious effects that falsely lower the A260/280 ratio and increases the sensitivity of the spectrophotometric detection of proteins contaminating nucleic acid preparations.

APPLICATION
Dilute the Phosphate Buffer stock solution with water to make a working solution of 1 - 3 mM Na2HPO4. The pH of 3 mM Na2HPO4 is about 8.4 - 8.6. Use the working solution to dilute aliquots of RNA or DNA for spectrophotometric analysis. Perform spectrophotometry according to standard procedures, taking care to use appropriate blank controls. Water treated with Phosphate Buffer is used to dilute RNA or DNA solubilized in any solution (water, FORMAzol® (cat.no. FO 121), SDS, or TE).

SPECIFICATIONS
RNase-free.
Molecular Biology Grade.
0.3 M Na2HPO4

PRODUCT INFORMATION
The ratio of the absorbance at 260 and 280 nm of nucleic acid solutions is commonly used to assess the purity of RNA and DNA preparations, although some reports have raised concerns about the accuracy and reliability of such measurements (1-4). In some cases, investigators contacting Molecular Research Center have obtained total RNA preparations that performed well in experiments, but which displayed low ratios upon spectrophotometric analysis. Also, scientists at Molecular Research Center have observed that simply changing the source of water used to dilute samples for spectrophotometry alters the A260/280 ratio. Subsequently, we performed a series of experiments examining the "Effect of pH and Ionic Strength on the Spectrophotometric Assessment of Nucleic Acid Purity"(5). The results of these experiments have practical applications regarding standard molecular biology procedures.

To summarize, the results show that the pH and ionic strength of water used to dilute RNA and DNA samples for spectrophotometric analysis significantly affect the A260/280 ratio. The pH is especially important and an acidic pH significantly decreases the A260/280 ratio of RNA and DNA. For example, a single RNA preparation exhibited an A260/280 ratio of 1.51 and 1.82 when measurements were performed in water with a pH of 6.1 and 8.0, respectively. Since the pH of distilled water is frequently between 5.0 - 6.0, this may be a key extraneous factor that falsely lowers A260/280 ratios of RNA preparations. We also observed that ionic strength of the diluent used in spectrophotometry may affect the ratio of RNA and DNA. Since most measurements of nucleic acids are performed in water, this second factor is of less importance. Based on these experiments, the spectrophotometric examination of RNA and DNA is most reliable when performed at pH 7.5 - 8.6. According to our observations, a slightly alkaline pH also increases sensitivity of the spectrophotometric detection of protein contamination in RNA and DNA preparations.

In light of these observations, Molecular Research Center has introduced the Phosphate Buffer as a solution that increases water pH to 8.4 - 8.6 and improves spectrophotometric measurements of nucleic acids. In addition, the use of Phosphate Buffer increases sensitivity of the spectrophotometric detection of proteins contaminating nucleic acid preparations. RNA and DNA preparations may be solubilized in water or any solution of your choice, including FORMAzol®, SDS or TNE buffer.

REFERENCES
  1. Glasel, J A. 1995. Validity of nucleic acid monitored by 260nm/280nm absorbance ratios. Biotechniques 18, 62-63. 1995.
  2. Huberman, J A. 1995. Importance of measuring nucleic acid absorbance at 240nm as well as at 260 and 280nm. Biotechniques 18, 636.
  3. Manchester, K L. 1995. Value of A260/A280 ratios for measurement of purity of nucleic acids. Biotechniques 19, 208-209.
  4. Manchester, K L. 1996. Use of UV methods for the measurement of protein and nucleic acid concentrations. Biotechniques 20, 968-970.
  5. Wilfinger, W W, Mackey, K and Chomczyski, P. 1997. Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques 22, 474-481.

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Last modified: February 16, 2011