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.
In this report, we present DNAzol, a patent-pending DNA isolation reagent containing guanidine thiocyanate and a detergent mixture. It is a complete, nontoxic and ready-to-use reagent for the isolation of genomic DNA from various biological sources. In the DNAzol protocol, a biological sample is homogenized (or lysed) in DNAzol, and the DNA is precipitated with ethanol, washed and dissolved in 8 mM NaOH. Following pH adjustment, the DNA can be used immediately for analysis or stored at 4 degrees C. The entire isolation can be completed in 20-30 min, and a wide range of DNA molecules can be isolated including genomic DNA and DNA fragments down to 0.1 kb in length. If necessary, samples can be stored in DNAzol at room temperature for extended periods of time. The isolated DNA is ready for PCR, Southern blotting and other molecular biology applications without any additional purification.
Specific sequences in RNA preparations can be detected by blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (northern blotting); unfractionated RNA is immobilized by slot or dot blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Northern blotting differs from Southern blotting largely in the initial gel fractionation step. Because they are single‐stranded, most RNAs are able to form secondary structures by intramolecular base pairing and must therefore be electrophoresed under denaturing conditions if good separations are to be obtained. Denaturation is achieved either by adding formaldehyde to the gel and loading buffers or by treating the RNA with glyoxal and dimethyl sulfoxide (DMSO) prior to loading. The describes blotting and hybridization of RNA fractionated in an agarose‐formaldehyde gel. Alternate protocols describe the glyoxal/DMSO method for denaturing gel electrophoresis and slot‐blot hybridization of RNA samples. Stripping hybridization probes from blots can be done under three different sets of conditions; these methods are outlined in a .
Specific sequences in RNA preparations can be detected by blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (northern blotting), while unfractionated RNA is immobilized by slot or dot blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Included in this unit are detailed procedures for RNA denaturation, blotting and hybridization. Also described is a method for stripped hybridization probes from blots so the blots can be re-hybridized with a different probe.
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