Protocol

Purification of Nucleic Acids by Extraction with Phenol:Chloroform

This protocol was adapted from "Commonly Used Techniques in Molecular Cloning," Appendix 8, in Molecular Cloning, Volume 3, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001.

INTRODUCTION

This protocol describes the standard method for nucleic acid purification by extraction first with phenol:chloroform (optionally containing hydroxyquiniline at 0.1%) and then with chloroform to remove any remaining phenol. The procedure takes advantage of the fact that deproteinization is more efficient when two different organic solvents are used instead of one.

MATERIALS

Reagents

Chloroform

Ethanol

Ether (optional)

Nucleic acid solution to be purified

Phenol:Chloroform (1:1)

TE (pH 7.8) (optional)

Equipment

Automatic pipettor fitted with a disposable tip

Pipettes, large-bore (optional)

Polypropylene tube

Rotating wheel (optional)

METHOD

1. Transfer the nucleic acid sample to a polypropylene tube and add an equal volume of phenol:chloroform.
   

   The nucleic acid will tend to partition into the organic phase if the phenol has not been adequately equilibrated to a pH of 7.8-8.0.
   

2. Mix the contents of the tube until an emulsion forms.
   
   
3. Centrifuge the mixture at 80% of the maximum speed that the tubes can bear for 1 minute at room temperature. If the organic and aqueous phases are not well separated, centrifuge again for a longer time.
   

   Normally, the aqueous phase forms the upper phase. However, if the aqueous phase is dense because of salt (>0.5 M) or sucrose (>10%), it will form the lower phase. The organic phase is easily identifiable because of the yellow color contributed by the 8-hydroxyquinoline that is added to phenol during equilibration.
   

4. Use a pipette to transfer the aqueous phase to a fresh tube. For small volumes (<200 µl), use an automatic pipettor fitted with a disposable tip. Discard the interface and organic phase.
   (see Troubleshooting)
   
   
5. Repeat Steps 1-4 until no protein is visible at the interface of the organic and aqueous phases.
   
   
6. Add an equal volume of chloroform and repeat Steps 2-4.
   
   
7. Recover the nucleic acid by standard precipitation with ethanol (see Standard Ethanol Precipitation of DNA in Microcentrifuge Tubes).
   

   Occasionally, ether is used to remove traces of chloroform from preparations of high-molecular-weight DNA. The organic and aqueous phases may be mixed by vortexing when isolating small DNA molecules (<10 kb) or by gentle shaking when isolating DNA molecules of moderate size (10-30 kb). To avoid shearing when isolating large DNA molecules (>30 kb), mix the organic and aqueous phases by rotating the tube slowly (20 rpm) on a wheel. Use large-bore pipettes to transfer the DNA from one tube to another.
   

TROUBLESHOOTING

Problem: Poor yield of nucleic acid.

[Step 4]

Solution: The organic phase and interface may be "back-extracted" as follows: After the first aqueous phase has been transferred, add an equal volume of TE (pH 7.8) to the organic phase and interface. Mix well. Separate the phases by centrifugation as in Step 3. Combine this second aqueous phase with the first, and proceed to Step 5.