Chapter:1 Protocol:12 Removal of Ethidium Bromide from DNA by Extraction with Organic Solvents
CHAPTER 1 > PROTOCOL 12
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Protocol 12
Removal of Ethidium Bromide from DNA by Extraction with Organic Solvents
Ethidium bromide is removed from DNA by phase extraction with organic solvents.
MATERIALS
CAUTION: Please click for information about appropriate handling of materials.
RECIPE: Please click for components of stock solutions, buffers, and reagents.
Buffers and Solutions
Ethanol
Isoamyl alcohol, saturated with H
2
O
n-Butanol, saturated with H
2
O
Phenol
Phenol:chloroform (1:1 v/v)
TE (pH 8.0)
Nucleic Acids
DNA sample, purified through CsCl gradient
Use material from either Step 7 of
Chapter 1, Protocol 10 or Step 5 of Chapter 1, Protocol 11.
METHOD
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.
6. Remove the CsCl from the DNA solution by ethanol precipitation (please follow Steps 7 through 12), by spin dialysis
through a microconcentrator (Amicon), 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.
7. To precipitate the DNA from the CsCl-DNA solution, measure the volume of the CsCl solution, add three volumes of
H
2
O, and mix the solution well.
8. Add 8 volumes of ethanol (1 volume is equal to that of the CsCl-DNA solution prior to dilution with H
2
O in Step 7) to the
DNA solution and mix well. Store the mixture for at least 15 minutes at 4°C.
9. Collect the precipitate of DNA by centrifugation at 20,000g (13,000 rpm in a Sorvall SS-34 rotor) for 15 minutes at 4°C.
10. 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 4°C and then collect the precipitate of DNA by centrifugation at 20,000g (13,000
rpm in a Sorvall SS-34 rotor) for 15 minutes.
11. 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.
12. Dissolve the precipitated DNA in 2 ml of H
2
O or TE (pH 8.0).
13. 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.
14. Measure the OD
260
of the final solution of DNA, and calculate the concentration of DNA. Store the DNA in aliquots at -
20°C.
REFERENCES
1.
Cozzarelli N.R., Kelly R.B., and Kornberg A. 1968. A minute circular DNA from Escherichia coli 15. Proc. Natl. Acad.
Sci. 60:992-999.
2. Wang J.C. 1969. Variation of the average rotation angle of the DNA helix and the superhelical turns of covalently
closed cyclic
DNA. J. Mol. Biol. 43:25-39.
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