Ethidium bromide solution

Ethidium bromide solution is supplied from many manufactures (e.g., AMRESCO, Ohio, Code No. E406). 

After electrophoresis, transfer the gel into ethidium bromide solution (50 pi stock solution in 1 1 of tap water) and stain for 5 -10 min. Transfer the gel onto a transilluminator and visualise the DNA with UV light, taking care to protect the skin and eyes against UV radiation. Document the result with a standard photograph or digital image analyser. 

Standardize the spectrofluorimeter in the following way. Pipet 2.0 mL of the pH 7.5, ethidium bromide-Tris buffer into a cuvette. Add 1.0 mL of Tris buffer I and 20 fiL of standard DNA solution. Mix and place in the fluorimeter. Adjust the fluorescence intensity to 100. Clean the cuvette as described in part A and repeat the assay using various concentrations of spermine. Prepare a table displaying the amount of each component to be added. Four reagents must be in the table pH 7.5, ethidium bromide-Tris buffer, DNA solution, spermine, and Tris buffer I. Maintain the volume of DNA at 20 fiL and ethidium bromide solution at 2.0 mL for all assays. Use 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of spermine in the assays. Remember that the total volume of all constituents in the cuvette must remain constant at 3.02 mL for all the assays. Therefore, the amount of Tris buffer I must change with the amount of spermine added. Prepare each assay separately by adding the proper amount of each component to the cuvette. Mix well and record the fluorescence intensity of each cuvette. 

Agarose slurry buffer, 0.04 M Tris-acetate, 0.002 M EDTA, pH 7.8 Ethidium bromide solution, 10 mg/mL in H20... 

After the agarose solution has cooled to 50°C, add ethidium bromide solution to give a final dye concentration of 0.5 p,g/mL. 

Ethidium bromide is a mutagen and a potential carcinogen. Always wear gloves and a face mask while weighing or handling the pure chemical. Gloves should always be worn while using solutions of the dye Do not allow the solutions of ethidium bromide to come into contact with your skin. Do not mouth pipet any ethidium bromide solutions. Save all assay mixtures and excess ethidium bromide solutions after measurements are taken, and pour them into a container marked Waste Ethidium. ... 

Prepare an ethidium bromide solution (50 pM) in a fluorescence cuvette and then plot both absorption and emission spectra (Xex = 470 nm). The molar extinction coefficient of ethidium bromide at 480 nm is 5680 M-1 cm-1. 

Finally, one can see that upon addition of DNA to the ethidium bromide solution, the orange color of the fluorophore turns to pink (Figure 12.4). This color modification is the result of the interaction between the two molecules and mainly binding of ethidium bromide to DNA. 

Visualize the DNA band with a long-wavelength UV lamp. Remove the DNA to 5-ml heat seal tubes, fill the tubes with CsCl-ethidium bromide solution (1.55 g/ml) and reband at 58,000 rpm for 6 hr or 44,000 rpm for 12 hr at 20° in a VTi 65 rotor (Beckman). 

Pour 1 ml of ethidium bromide solution (1 pg/ml) on top of the gel. Cover with plastic wrap or with a plastic plate transparent to ultraviolet light. 

The DNA can he seen directly in the CsCl gradient by reversibly staining with ethidium bromide (Firtel and Bonner 1971). Solid CsCl is added to the DNA solution to make the density up to about 1.55 g ml (0.97 g CsCl to each 1 ml original DNA solution) followed hy 1/20 volume of ethidium bromide solution (10 mg/ml in water). Spinning time is 48 hr at 40,000 rev/min. RNA and... 

Ethidium bromide solution 10 mg mH stock solution made up of 100 mg of ethidium bromide and 10 ml of water... 

Storage and Handling Ethidium bromide should be handled in the laboratory using the "basic prudent practices" described in Chapter 5.C. Because of its mutagenicity, stock solutions of this compound should be prepared in a fume hood, and protective gloves should be worn at aU times while handling this substance. Operations capable of generating ethidium bromide dust or aerosols of ethidium bromide solutions should be conducted in a fume hood to prevent exposure by inhalation. 

Ethidium Bromide and a 2.0 1 dC-dG Ethidium Bromide Solution (25 C). The concentration of ethidium bromide was 1.6 ieM in a 5 inM D2O phosphate buffer (pD - 7.4) in each spectrum. EthBr, ethidium bromide, TSP, sodium 3-trimethyls ilylpropionate-2, 2,3,3-di. ... 

CAUTION ethidium bromide is a powerful mutagen Gloves should be worn when handling solutions of this dye and stained gels. Decontamination of ethidium bromide solutions is described in Sambrook etal (6) page 6.16-6.17. 

Add 0.8 ml ethidium bromide solution and mix immediately. Wrap the tube in silver foil.f Give a quick spin at 8000 r.p jn. for 5 min and immediate transfer the dear, red solution under the scum to a Beckman Quick-Seal tube using a 10 ml disposable syringe fitted with an 18 gauge hypodermic needle. Tare tubes as predsely as possible. Rll the remaining of the tube with mineral oil and seal tubes. Avoid bubbles at the top. 

Pour the ethidium bromide solution back into its storage bottle. Pour eitough wrater into the staining tray to cover the gel. Wait 5 minutes. Pour the water out of the staining tray into a hazardous waste contairrer and place the... 

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