Detection with ethidium bromide

Visual examination of crystals using a light microscope does not indicate whether the crystals consist of only the protein or the protein-DNA complex. Therefore, the crystals are washed free of any uncrystallized DNA and protein several times with a solution containing the precipitant and any additives, etc. at the concentration and pH used for growing crystals (mother liquor). Finally, the crystals are separated from the mother liquor by microcentrifugation, dissolved in a suitable buffer, and analysed biochemically. The protein content is determined by SDS polyacrylamide gel electrophoresis, the protein concentration by BIO-RAD assay, and amino acid composition by mass-spectroscopy. The DNA can be detected by staining the gel with ethidium bromide or methylene blue (Jordan et al., 1985), whereas... 

Matsuno Y, Kinoshita M, Kakehi (2005) Fast analysis of glycosaminoglycans by microchip electrophoresis with in situ fluorescent detection using ethidium bromide. J Pharm Biomed Anal 37 429-436... 

An important development in CE technology that has helped to promote the analysis of PCR products by CGE is the introduction of laser-induced fluorescence (LIF) detection [4-7]. Because LIF can increase the sensitivity of detection for dsDNA by more than 400-fold over UV detection, it has become the method of choice for the vast majority of dsDNA separations [5]. A practical illustration of the advantage of LIF detection is that typical separations of PCR products by slab-gel electrophoresis with ethidium bromide staining require approximately 5 ng of DNA per band for adequate detection, whereas, with CGE-LIF, sub-picogram levels of DNA are readily detected [6]. 

After amplification, tlie products can be detected by various methods. Simple gel electrophoresis with ethidium bromide staining may suffice. When greater accuracy is required, one of the primers can be fluorescently labeled so that after PCR the fragments are accurately sized on a DNA sequencing device. Alternatively, some form of hybridization assay can be used to verify or analyze the amplified product. Automated methods are always attractive and closed-tube methods are particularly advantageous in the clinical laboratory. Adding a fluorescent dye or probe before amplification allows thermocyclers equipped with optical detection to analyze the reaction as it progresses (real-time PCR) or after the reaction is complete (endpoint measurement) without need to process the sample for a separate analysis step. 

The first detection methods used with PCR were radioactively labeled probes to identify specific amplified sequences (M8, SI). With improvements in amplification specificity it became possible to visualize amplified DNA of the predicted size directly by its fluorescence on an agarose or polyacrylamide gel (M9) following staining with ethidium bromide. Probe-based methods remain a key feature of current detection systems primarily because of the additional information and sequence specificity they provide. Probes have been converted to nonisotopic colorimetric systems (B6) by labeling them with an enzyme such as... 

The presence of amplified PCR products was detected in two ways. Ethidium bromide included in the reaction mixture allowed rapid determination of PCR products in the capillaries by visualization of the capillaries under UV illumination (see Note 9). After that determination, the reaction was passed through a 1.5% agarose gel by electrophoresis in lx TBE buffer. Gels were stained with ethidium bromide and photographed in UV light with a Kodak digital camera EDAS 290. 

Fig. 5 Reverse transcriptase-PCR analysis of NnAChR messenger RNA (mRNA) in rat vagal pulmonary sensory neurons. Cytoplasm of 20 isolated pulmonary sensory neurons was collected into a single PCR tube. One-step Reverse transcriptase-PCR and a nested PCR were carried out to detect the presence of nicotinic mRNA subunits. The nested PCR products were run on a 1% agarose gel and stained with ethidium bromide. M 100-bp DNA marker. (From Gu et al. 2008, with permission)...

For DNA detection, the gels are washed with water and stained with ethidium bromide. Polaroid photographs are taken under UV transillumination at 254 nm. Visual examination of the gels is sufficient to get useful information about DNA stability. Densitometric analysis can be used to determine approximately the relative amount of the different DNA forms present in the gel. However, such determination is not very precise at high DNA concentrations, since labeling with ethidium bromide does not increase linearly with DNA under these conditions. For accurate results, it is necessary to radioactively label DNA and use a Phospholmager. 

As the salt concentration increased, the electrostatically bound acridine orange was displaced from the DNA, decreasing the observed polarization value. As shown in figure 2, at low concentrations of ethidium bromide, the initial polarization value decreased with increasing salt concentration, and remained relatively constant at low ethidium bromide concentrations. Once the detection limit is reached, the polarization decreases linearly with ethidium bromide concentration. In figure 3, a normalized version of figure 2, the detection limit (LOD), the point where the polarization starts to decrease, is shown to be unaffected by salt concentration. 

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