Experimental Principle
The separation of plasmid and chromosome DNA by suspension density gradient centrifugation containing cesium chloride and ethidium bromide depends on the difference in the amount of ethidium bromide bound to linear DNA and closed-loop DNA. Ethidium bromide binds to DNA by embedding between bases, thus unwinding the double helix, resulting in an increase in the length of the linear DNA. As compensation, superhelix units will be introduced into the closed-loop plasmid DNA. Finally, the superhelicity is greatly increased, thus preventing the continued intercalation of ethidium bromide molecules. However, linear molecules are not subject to this limit and can continue to bind more ethidium bromide until they reach saturation (about 1 ethidium bromide molecule for every 2 base pairs). Due to the difference in the binding amount of dyes, linear and closed-loop DNA have different buoyancy densities in cesium chloride containing saturated ethidium bromide, thus purifying DNA.
Schematic diagram of density gradient centrifugation.
Product List
Click the product name to view product details.
| CAS NO. | Product Name | Inquiry |
| 206444-57-7 | Ethidium bromide-N N-bismethacrylamide | Inquiry |
| 480438-67-3 | Ethidium bromide-N N-bisacrylamide | Inquiry |
| 3,8-Dimethacryloyl ethidium bromide | Inquiry | |
| 58880-05-0 | Ethidium monoazide bromide | Inquiry |
| 7647-17-8 | Cesium Chloride | Inquiry |
| 15844-58-3 | Cesium chloride/sodium chloride(2:1)e& | Inquiry |
| 7647-14-5 | Sodium chloride | Inquiry |
| 7647-15-5 | Sodium Chloride | Inquiry |
| 61990-73-6 | N-Butanol,[1-14c] | Inquiry |
Experimental Reagents
1. CsCl (solid)
2. CsCl rezoning solution
3. Ethanol
4. Ethidium bromide (10 mg/mL)
5. Sodium chloride
6. n-butanol
7. TE buffer
8. Nucleic acid and oligonucleotide
9. Crude plasmid
Experimental Procedure
1. Dissolve the precipitate obtained in the last step of the crude lysate preparation protocol in 4 mL TE buffer, add 4.4 g CsCl, and then add 0.4 mL 10 mg/mL ethidium bromide after dissolution. If using the supernatant, scale up the CsCl and ethidium bromide proportional to the volume of the supernatant.
2. Transfer the solution into a 5 mL quick-seal ultracentrifuge tube, add 1 g/mL CsCl/TE solution to the tube, and seal the centrifuge tube.
3. Centrifuge at 500,000 g for 3.5 hours at 20 °C or 350,000 g for 14 hours or more at 20 °C.
4. Carefully remove the tube, insert a 20-G needle from the top of the tube, and aspirate the plasmid band using a 3 mL syringe with another 20-G needle (the needle is inserted approximately 1 cm below the plasmid band).
5. In order to obtain higher purity plasmid DNA, perform a second ultracentrifugation in a new tube with CsCl/TE buffer containing 0.1 mg/mL ethidium bromide on top of the tube.
6. Fill the Dower AG50W-X8 column with 1.5 to 2 times the volume of the plasmid DNA/EB solution, and wash and balance the column with several times the volume of TE/0.2 mol/L NaCl solution.
7. Use a syringe to add the plasmid DNA solution mixed with ethidium bromide directly to the top of the resin, being careful not to shake the resin. Collect the effluent immediately.
8. Elute the column twice with a 0.2 mol/L NaCl/TE solution that is twice the volume of the loading plasmid solution, and collect the effluent in the same tube.
9. Ethidium bromide can also be removed with n-butanol saturated with equal volume TE. Fully swirling and mixing, then removing the organic phase, repeating several times until no red is seen under the UV lamp, and then adding 3 times the volume of TE buffer to dilute cesium chloride, the organic waste containing ethidium bromide produced in this method should be properly disposed of.
10. Add 2 times the volume of 100% ethanol to precipitate plasmid DNA at room temperature or -20 °C. Centrifuge at 10,000 g for 10 min at 4 °C. When precipitating DNA, the temperature should not be lower than -20 °C to avoid precipitation of cesium chloride.
11. The precipitate was washed once with 70% ethanol, dried under vacuum, dissolved in TE buffer, and stored at 4 °C.
Precautions
1. Ethidium bromide is a mutagen and can cause environmental pollution. Gloves should be worn when operating and handled properly.
2. Centrifugation must be performed at a temperature of no less than 15 °C to prevent damage to the rotor and centrifuge and potentially serious injury to personnel.
3. To prevent serious damage to your eyes from ultraviolet rays, you should wear UV protective glasses or a mask. Wear gloves when handling ethidium bromide.
