Ethidium bromide staining

Cariello, N., Keohavong, P., Sanderson, B., Thilly, W. DNA damage produced by ethidium bromide staining and exposure to ultraviolet light. Nucleic Acids Research, Vol. 16, No.9, (May 1988), pp. 4157-4161, ISSN 0305-1048... 

The final concentration of the cRNA transcript can be determined by its absorbance. The absorbance of a 1 1000-2000 dilution of the transcript is read at a wavelength of 260 nm (one unit A260 is equivalent to 40 pg/mL of RNA). Alternatively, densitometry on ethidium bromide-stained agarose gels or colorimetric stains (e.g., RiboGreen, Molecular Probes, Inc., Eugene, OR) can be used to... 

Perform electrophoresis, and visualize the mRNA sample by ethidium bromide staining. If the RNA band is smeared or not visible, possible causes may be degradation of mRNA by RNase contamination. 

Nucleic acids can be visualized by ethidium bromide staining, UV shadowing, or phosphorimaging of radioactive samples. A sterile scalpel should be used to excise the separated product which can then be eluted by electrophoresis (1 x Tris-borate, pH 8.3) into a 30kDa molecular weight cutoff (MWCO) filter (Millipore). The product is concentrated by centrifugation, dialyzed, and resuspended in the buffer of choice. The yield of nucleic acid is typically 75 %, and ethanol precipitation is not needed. 

Figure 1.3 At left is a schematic of the PCR-based method of generating DNA templates for in vitro transcription. The three-reaction method yields the DNA template shown at the bottom. Transcription from this DNA results in the desired RNA product linked to the affinity purification tag. At right is an ethidium bromide stained agarose gel showing representative results from reactions la, lb, and 2. Reprinted with permission from Edwards et al. (2009).

Kido, N., Ohta, M., Kato, N. Detection of lipopolysaccharides by ethidium bromide staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J Bacteriol 172 (1990) 1145-1147. 

Make a dilution of the unknown DNA that falls in the range 10-100 ng//d. An estimation of the initial concentration of each unknown can be made by either ethidium bromide staining on an agarose gel, a preliminary run-through of the densitometric procedure, or the knowledge of the yield of DNA usually obtained from the source material. 

Photograph the microcapillaries much as you would an ethidium bromide-stained gel, but use f 8 or higher rather than a lower f-stop. This reduces lens distortion and gives sharper images. The frame should allow a generous border around the microcapillary array for the same reason do not fill the frame with arrays. Record all camera settings exactly (f-stop,... 

Although the primers used in this technique are random sequences, there are several criteria that must be met to produce results. Williams et al found that the minimum primer length to detect amplification in ethidium bromide-stained agarose gels was 9 bases, and a minimum of 40% GC content was also required to produce amplification (50-80% GC content is generally used). These criteria were determined at an annealing temperature of 36° but also held true at annealing temperatures as low as 15°. 

Fig. 2. Effect of DNA concentration and number of PCR cycles on RAPD analysis, shown on ethidium bromide-stained agarose gels, using template DNA from a single individual titmouse (Parus bicolor) and the 10-base primer AP5a4 (5 CTGTTGCTAC 3 ). (A) Various concentrations of template DNA amplified through 45 cycles of PCR. Lanes 1-11 contain 100, 50, 20, 10, 5, 2, 1, 0.1, 0.05, 0.01, and 0.005 ng of template DNA, respectively, in 25-/

As compared to ethidium bromide-stained agarose gels, many more amplified bands can be visualized by separating die products of the reaction on a polyacrylamide gel and staining with silver.9 This gel system also permits detection of bands amplified weakly by primers as short as 5 nucleotides. 

Fig. 3. RAPD analysis on an ethidium bromide-stained agarose gel using 0.6 ng//

Cleave several -fig aliquots of the DNA with different restriction endonucleases, following the manufacturer s recommendations. Fractionate the digested DNA by electrophoresis through 0.8% (w/v) agarose gels, then visualize the DNA by ethidium bromide staining and transillumination. 

To facilitate cloning and to increase the probability that all variants will be represented, amplifications should be as specific and efficient as possible. Ideally, the amplified product should be visible by ethidium bromide staining of agarose gels. 

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