DNA提取中EB的去除实验方法

• Ethanol

• Isoamyl alcohol or n-butanol, saturated with H₂O
  One or the other of these organic solvents is required to remove ethidium bromide from the DNA preparation after centrifugation.

• Phenol
  Optional, please see Step 13.

• Phenol:chloroform (1:1 v/v)
  Optional, please see Step 13.

• TE (pH 8.0)

• DNA sample, purified through CsCl gradient

• Sorvall RT-6000 centrifuge with an HL-4 rotor and 50-ml buckets or equivalent

• Sorvall SS-34 rotor or equivalent

• Dialysis tubing and clips
   or 
  Equipment for spin dialysis through a microconcentrator
  Optional, please see Step 6.

Extraction of DNA Solution to Remove Ethidium Bromide

1. To the solution of DNA in a glass or polypropylene tube, add an equal volume of either water-saturated n-butanol or isoamyl alcohol. Close the cap of the tube tightly.
    
   

2. Mix the organic and aqueous phases by vortexing.
    
   

3. Centrifuge the mixture at 450g (1500 rpm in a Sorvall RT-6000 centrifuge with an HL-4 rotor and 50-ml buckets) for 3 minutes at room temperature or stand the solution at room temperature until the organic and aqueous phases have separated.
    
   

4. Use a Pasteur pipette to transfer the upper (organic) phase, which is now a beautiful deep pink color, to an appropriate waste container.
    
   

5. Repeat the extraction (Steps 1-4) four to six times until all the pink color disappears from both the aqueous phase and organic phases.
   

Removal of CsCl from the DNA Solution

1. Remove the CsCl from the DNA solution by ethanol precipitation (please follow Steps 7 through 12), by spin dialysis through a microconcentrator, or by dialysis overnight (16 hours) against 2 liters of TE (pH 8.0) (change buffer frequently). If one of the latter two methods is used, then proceed to Step 13.
    
   

2. To precipitate the DNA from the CsCl-DNA solution, measure the volume of the CsCl solution, add three volumes of H₂O, and mix the solution well.
    
   This addition dilutes the CsCl and prevents precipitation of the salt by ethanol. 
    
   

3. Add 8 volumes of ethanol (1 volume is equal to that of the CsCl-DNA solution prior to dilution with H₂O in Step 7) to the DNA solution and mix well. Store the mixture for at least 15 minutes at 4ºC.
    
   Higher recoveries of DNA can be realized if the precipitation reaction is allowed to occur overnight at 4ºC. 
    
   IMPORTANT CsCl precipitates if the ethanolic solution of DNA is stored at -20ºC. 
    
   

4. Collect the precipitate of DNA by centrifugation at 20,000g (13,000 rpm in a Sorvall SS-34 rotor) for 15 minutes at 4ºC.
    
   

5. Decant the supernatant to a fresh centrifuge tube. Add an equal volume of absolute ethanol to the supernatant. Store the mixture for at least 15 minutes at 4ºC and then collect the precipitate of DNA by centrifugation at 20,000g (13,000 rpm in a Sorvall SS-34 rotor) for 15 minutes.
    
   Not all of the plasmid DNA is recovered in the first precipitation, hence the addition of a second batch of ethanol (Hildeman and Muller 1997). 
    
   

6. Wash the two DNA precipitates with 70% ethanol. Remove as much of the 70% ethanol as possible and then allow any remaining fluid to evaporate at room temperature.
    
   

7. Dissolve the precipitated DNA in 2 ml of H2O or TE (pH 8.0).
    
   For DNA sequencing, the DNA should be dissolved in H₂O. TE (pH 8.0) is a better option if the DNA is to be stored for a long period of time. 
    
   

8. If the resuspended DNA contains significant quantities of ethidium bromide, as judged from its color or its emission of fluorescence when illuminated by UV light, extract the solution once with phenol and once with phenol:chloroform, and then again precipitate the DNA with ethanol.
    
   

9. Measure the OD₂₆₀ of the final solution of DNA, and calculate the concentration of DNA. Store the DNA in aliquots at -20ºC.