Ethidium bromide and

DNA molecules on gel are stained with ethidium bromide and observed using epi-fluorescent microscopy. 

Intercalating agents are hydrophobic, planar structures that can fit between the DNA base pairs in the center of the DNA double helix. These compounds (ethidium bromide and actinomycin D are often-used examples) take up space in the helix and cause the helix to unwind a little bit by increasing the pitch. The pitch is a measure of the distance between successive base pairs. 

Make a special note when handling strong acids or bases, phenol, ethidium bromide and acrylamide — the main chemicals. 

Fig. 6. Apoptotic DNA ladder pattern of eosinophils treated with dexamethasone (Dexa, 2 (xM) for 18 h (Zl). DNA was extracted from cells with ethanol (P4) and electrophoresed on 1% agarose gel in 1 X TAE (Tris acetate-EDTA) buffer (pH 8.0). After electrophoresis, the gel was soaked in 1 x TAE buffer containing 0.5 /tg/ml ethidium bromide, and DNA was visualized by an ultraviolet illuminator. Reproduced with permission from Zhang, J. R, Wong, C. K., Lam, C. W. K., Ho, C. Y., and Hjelm, N. M., Biochemical assessment of apoptosis. Chinese J. Lab. Med. Clin. Sci. 1, 27-28 (2000).

Volatile buffers were reconsidered for the modified method. Triethylamine was ruled out primarily because it could not be obtained in high purity and because the secondary and primary amines contaminating it could potentially react with solutes present in the water sample. Preliminary evidence of reaction between ethidium bromide and triethylammonium bicarbonate was obtained, but the reaction product was not characterized. The components of volatile buffers that appeared acceptable on the basis of chemical purity were ammonia, acetic acid, and formic acid. A few exploratory experiments were conducted involving the elution by ammonium formate and ammonium acetate of EB or quinaldic acid exchanged onto AG MP-50 or IRA 900. These experiments showed that 1 M ammonium formate in water was a very poor eluent, but that EB could be eluted from AG MP-50 with 1 M ammonium formate in methanol. Elution was essentially complete with 6 bed volumes of the methanolic eluent, whereas neither methanol alone nor aqueous 1 M ammonium formate was able to elute this solute. This situation pointed out the necessity for a counterion to displace exchanged solutes and, additionally, indicated that the displaced solute be highly soluble in the eluting solvent. 

J. Le Pecq and C. Paoletti,/. Mol. Biol. 27, 87 (1967). A Fluorescent Complex Between Ethidium Bromide and Nucleic Acids. ... 

Unlike FACS, cell viability can be measured during the separation procedure using ethidium bromide and acridine orange... 

Ethidium bromide/acndine orange stock solutions consist of 50 mg of ethidium bromide and 15 mg of acridine orange, dissolved in 1 mL of 95% ethanol and made up to 50 mL with distilled water. Store at -20°C. 

For use in viability counts, stocks are diluted 1.100 with PBS. Stored at room temperature in the dark. The solution has a shelf-life of at least 2 mo. Hazard warning both ethidium bromide and acridine orange have mutagenic properties, are combustible, toxic, and irritants to the skin, eyes, and respiratory system. Handle with care... 

Template DNA for the production of hybridization probes must be pure Standard methods, such as dye-buoyant density ultracentnfugation, generate acceptable products. Ethidium bromide and cesium chloride are removed prior to use of the DNA (4)... 

Typical OH-scavengers suppress this reaction of (OP)2Cu+ (Que et al. 1980) yet, acetate and benzoate seem to be equally efficient, despite the fact that acetate is nearly two orders of magnitude less reactive towards OH than benzoate (k=7 X 107 dm3 mol-1 s 1 vs k = 5 X 109 dm3 mol-1 s 1 Buxton et al. 1988), and obviously it is not a freely diffusing OH that is responsible for the reaction. The reaction is also suppressed by Cu-complexing compounds and by transition metal ions such as Zn2+, Co2+, Cd2+ and Ni2+ that form stable complexes with 1,10-phenanthroline (Que et al. 1980) and also by competitive intercalators such as ethidium bromide and 2,9-dimethyl-l,10-phenanthroline (Reich et al. 1981). Interestingly, compared to its parent, the latter is inactive. NADH may serve as a reductant, but 02, seems to be a salient intermediate in this cleavage reaction, because cleavage is fully suppressed in the presence of SOD (Reich et al. 1981). 

Much attention has been focussed lately on the family of asymmetric cyanine dyes for use in fluorescence detection of nucleic acids. These dyes show a significant enhancement in fluorescence intensity (100- to 1000-fold) upon binding to double-stranded DNA as compared to that from the fluorophore in solution. Use of cyanine fluorophores may be advantageous for use in assay design and sensor applications with respect to some of the more commonplace dyes, such as ethidium bromide and Hoechst 33342, as these latter dyes exhibit significant fluorescence intensity as background when in solution and have significantly lower enhancement in emission intensity [42]. 

Intercalators associate with dsDNA by insertion between the stacked base pairs of DNA [52], EtBr binds to dsDNA with little to no sequence specificity, with one dye molecule inserting for every 4-5 base pairs [53]. It also binds weakly via a non-intercalative binding mechanism only after the intercalative sites have been saturated [54], Propidium iodide (PRO) is structurally similar to ethidium bromide, and both dyes show a fluorescence enhancement of approximately 20-30 fold upon binding to dsDNA [41]. As well, their excitation maxima shift 30-40 nm upon binding due to the environment change associated with intercalation into the more rigid and hydrophobic interior of the double-stranded nucleic acid structure relative to aqueous solution [41]. 

Starting with the first IPCR study, gel electrophoresis retains its potential as a fast and easy method for end-point determination of DNA amplificate for IPCR assays [10, 24, 25, 29, 31, 35, 36, 38, 39, 64], Readout is performed by intercalation fluorescence markers (e.g., ethidium bromide) and photometric/densitometric quantification of band signal intensities. The direct addition of a double-strand specific intercalation marker to the PCR amplificate and subsequent measurement of fluorescence in microwells proved to be of insufficient sensitivity for the quantification of IPCR amplificate [37]. Alternative approaches, such as radioactive labeling during PCR and subsequent imaging [33], were carried out but are not well suited for routine clinical application because of additional methodological requirements. An advantage of gel electrophoresis is the possibility of simultaneous amplificate detection for multiplex IPCR [41] and the ability to detect nonspecific amplification products. 

Clumps of suspension-adapted CHO cells stained with ethidium bromide and acridine orange, observed under a fluorescence microscope. Photo taken by Dr. Rodrigo C. V. Pinto, Cell Culture Engnieering Laboratory, Federal University of Rio de Janeiro, Brazil. 

Fig. 11 Lipid vesicles with encapsulated T7 RNA polymerase and DNA template. A mixture of four nucleoside triphosphates was added, and these diffused into the vesicles and were used by the polymerase to synthesize RNA with DNA as a template. The RNA was stained with ethidium bromide and appears as fluorescent material within the vesicles. Note that some of the vesicles do not contain fluorescent RNA, presumably because they lacked sufficient enzyme or template. Scale bar shows 20 pm...

Fig. 6.14. Agarose gel electrophoresis of Echinococcus granulosus (horse strain) RNA. The samples in lanes 1,2,7 and 16 were prepared in 10 mM phosphate buffer, pH 6.8, and were not denatured. The samples in lanes 3, 4, 8 and 13-15 were denatured in 50% (w/v) dimethylsulphoxide in 10 mM phosphate buffer, pH 6.8. The samples in lanes 5,6,9 and 10 12 were denatured in 1 m glyoxal/3 m urea in 10mM phosphate buffer, pH 6.8. Lanes 1,3 and 5, E. granulosus total nucleic acids (10 fig) lanes 2,4 and 6, E. granulosus RNA (5 fig) lanes 7,8 and 9, Escherichia coli RNA (10 fig) lanes 10-16, Schistosoma mansoni RNA (10 fig). The E. coli RNA was included as a molecular weight marker the larger subunit is approximately 1 000 000 and the smaller subunit is 500 000. The RNA was visualised by staining with ethidium bromide and ultraviolet illumination. (After McManus et al., 1985.)...

For the solutions containing Trp, riboflavin, ethidium bromide, and ANS alone, normalize the absorption and excitation spectra and superimpose them. We suggest that the normalization be carried out at 280, 450, and 280 nm, for L-Trp, riboflavin, and ANS, respectively. 

Since ANS dissolved in a polar medium does not fluoresce, one cannot record its fluorescence excitation spectrum. For tryptophan, ethidium bromide, and riboflavin, one can see that for each molecule, the absorption spectrum looks like the fluorescence... 

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