Ethidium bromide in DNA purified by cesium chloride gradient centrifugation can be removed by ion-exchange chromatography, followed by ethanol precipitation to remove cesium chloride. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Experimental removal of ethidium bromide from DNA by ion exchange chromatography
Principle
Ethidium bromide in DNA purified by cesium chloride gradient centrifugation can be removed by ion-exchange chromatography, followed by ethanol precipitation to remove the cesium chloride.
Materials and Instruments
DNA Sample Move I. Materials For more product details, please visit Aladdin Scientific website.
Ethanol HCl NaCl Phenol Chloroform TE TEN Buffer
Dowex AG50W-X8
1. Buffers and solutions
Ethanol, HCl (1 mol/L), NaCl ( 5 mol/L), phenol, phenol:chloroform (1:1, V/V), TE (pH 8.0), TEN buffer containing 0.2% sodium azide.
2. Nucleic acids and oligonucleotides
DNA samples purified by cesium chloride gradient centrifugation.
3. Centrifuge and rotor
Sorvall SS-34 rotor or equivalent.
4. Specialized equipment
Dowex AG50W-X8 (100~200 mesh, dry size) (Dowex AG50W-X8 is a cation exchange resin available from BioRad.), glass wool, refractometer (optional) (Although not required, a refractometer is useful in estimating the density of cesium chloride solution. (Dowex AG50W-X8 is a cation exchange resin available from BioRad.), glass wool, refractometer (optional) (Although not required, a refractometer is useful in estimating the density of cesium chloride solutions.) The methodology is summarized below.
II. Methods
1. Equilibrate Dowex AG50 resin before use:
(1) Dissolve about 20 g of Dowex AG50 resin in about 100 ml of 1 moI/L NaCI and stir for 5 min. After the resin sinks, aspirate the supernatant and discard.
(2) Add about 100 ml of 1 mol/L HCl and continue to stir the suspension for 5 min. After the resin sinks, aspirate and discard the supernatant.
(3) Repeat this procedure twice with water (100 ml each) and wash once with 100 ml of TEN buffer.
(4) Store the resin in TEN buffer containing 0.2% sodium azide at 4°C. The resin is then washed with 100 ml of TEN buffer.
2. Pack a 1 ml Dowex AG50 column in a Pasteur pipette as shown in the figure. 
3. Remove the buffer from the resin, wash the column with 2x the volume of TE (pH 8.0), and add the DNA containing ethidium bromide and cesium chloride directly to the resin.
4. Immediately begin collecting the effluent from the column. When all of the DNA solution has entered the column, elute the resin with 1.2 times the volume of TE ( pH 8.0) in the column bed and continue to collect the effluent in a 30 ml Corex tube.
5. After the column has run dry, dilute the effluent with 2.5 times the volume of water.
6. Add 8 times the volume of ethanol and leave at 4°C for 15 min to precipitate the DNA. The DNA is then collected by centrifugation at 17,000 g (12,000 r/min, Sorvall SS-34 rotor) for 15 min at 4 °C.
7. Pour the supernatant into a clean centrifuge tube, add an equal volume of anhydrous ethanol, allow to stand at 4°C for at least 15 min, and then centrifuge at 20,000 g (13,000 r/min, Sorvall SS-34 rotor) for 15 min to collect the DNA.
If the first precipitation does not yield all the plasmid DNA, add ethanol again (Hildeman and Muller 1997).
8. Wash the DNA precipitates obtained twice with 70% ethanol, then remove as much of the 70% ethanol as possible and allow the residual liquid to evaporate at room temperature.
9. Dissolve the DNA precipitate with 2 ml of H2O or TE (pH 8.0).
For DNA sequencing, the DNA should be dissolved in H2O; if the DNA is to be stored for a long time, TE (pH 8.0) is a better choice.
10. If it is clear that the resolubilized DNA still contains ethidium bromide, either by its color or by the fluorescence it gives off when exposed to ultraviolet light, extract the DNA once more with phenol or phenol:chloroform and then precipitate the DNA with ethanol.
11. Determine the OD260 value of the final DNA solution and calculate the concentration of DNA. Store at -20°C in small portions.