Phenol-chloroform extraction

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Phenol-chloroform extraction is a liquid-liquid extraction technique in biochemistry and molecular biology for isolating DNA, RNA and protein. where equal volumes of a phenol:chloroform mixture and the aqueous sample are mixed, forming a biphasic mixture. This method takes longer than a column-based system such as the RNeasy column (Qiagen), but has better purity and has the major advantage that no RNA is lost: an RNA column typically cannot adsorb RNA transcripts shorter than 200 bp, including many types of noncoding RNAs including siRNA and miRNA, hot research topics in modern molecular biology.

It was originally devised by Piotr Chomczynski and Nicoletta Sacchi and published in 1987 (referred to as Guanidinium thiocyanate-phenol-chloroform extraction).[1][2]

Contents

[edit] How it works

This method is widely used and is often referred to as the TRIzol method, after the name of the Invitrogen product. It relies on phase separation upon centrifugation of a mix of the aqueous sample and a solution contaning water-saturated phenol, chloroform and a chaotropic denaturing solution (guanidinium thiocyanate) with an upper aqueous phase and a precipitated organic phase (phenol). Nearly all of the RNA collects into the aqueous phase, while the DNA and protein collect in the interphase and the organic phase. This is then followed by precipitation with ethanol of the nucleic acids. The name of the protocol derives from the chemicals used: The proteins, including RNase, are denatured by guanidinium thiocyanate which is strong enough to separate rRNA from ribosomes. Other denaturing chemicals such as 2-mercaptoethanol and sarcosine are often present. Phenol, isopropanol and water are the solvents that mix poorly, and, in the presence of chloroform or BCP (bromochloropropane), separate entirely into two phases that are easily recognized by their color: the clear, upper water phase and the bright pink lower phase.

The major downside is that Phenol and chloroform are both hazardous and inconvenient materials, and the extraction is laborious, so in recent years many companies now offer alternative ways to isolate DNA.

[edit] Guide

[3][4]

[edit] Reagents

  • Phenol. The phenol used for biochemistry comes as a water-saturated solution with Tris buffer, as a Tris-buffered 50% phenol, 50% chloroform solution, or as a Tris-buffered 50% phenol, 48% chloroform, 2% isoamyl alcohol solution (sometimes called simply "25:24:1"). Phenol is naturally somewhat water-soluble, and gives a 'fuzzy' interface that is sharpened by the presence of chloroform. The isoamyl alcohol reduces foam, which is a problem with phenol:chloroform. Most solutions also have an antioxidant, as oxidized phenol will damage the DNA. Pure phenol crystals are no longer common. These had to be equilibrated into the buffer and then melted and dissolved, with care taken to avoid inhalation of the fumes or fine aerosolized powders.
  • Chloroform. The chloroform will be stabilized with small quantities of amylene or ethanol because storage of pure chloroform solutions in oxygen and ultraviolet light will produce phosgene gas. Chloroform sometimes also comes as a 96% chloroform, 4% isoamyl alcohol solution that can be mixed with an equal volume of phenol to make 25:24:1.
  • Isoamyl alcohol. Whether to purchase isoamyl alcohol separately or mixed in with the phenol and chloroform stocks is largely an individual choice, although it is sensible to coordinate with the rest of the lab so reagents can be shared. Some protocols do not require it at all.
  • For RNA extraction, use Propan-2-ol (isopropanol) in lieu of Isoamyl alcohol.

[edit] Protocol

Specific protocols vary between labs and even individual workers. A sample is given below. Note that a "volume" means an amount equal to the volume of the original DNA sample, e.g., 100μL is one volume for a 100μL sample.

  1. Put 100-700μL of sample into a 1.5mL microcentrifuge tube.
    • Use water to dilute samples smaller than 100μL. The aqueous phase must be thick enough to see and remove, which is difficult for volumes less than 100μL. After ethanol precipitation, the DNA pellet can be resuspended at any desired concentration.
    • Divide samples larger than 700μL into multiple tubes. A sample larger than 700μL will not fit in the tube with a volume of phenol. If this is inconvenient, protocols exist that use tubes as large as 50mL, with special precautions taken to ensure even mixing.
  2. Add an equal volume of phenol to the tube.
    • Phenol:chloroform:isoamyl alcohol will give a sharper interface, and 25:24:1 will show less foam.
    • Although pipettes and micropipette tips are usually resistant, phenol is known to attack polycarbonate plastic (clear and hard). For phenol:chloroform mixtures or for chloroform, glass pipettes should be used, or micropipettors exclusively, as the chloroform is usually able to attack plastic pipettes. In general, work as quickly as possible without sacrificing accuracy.
  3. Vortex vigorously to mix the phases.
    • Small plasmids can withstand vigorous vortexing. It is even safe, although usually unnecessary, to hold two tubes together in the vortex cup so they collide violently during the vortexing. However, as the length of the DNA increases, so does the risk of shearing. Inverting 5-10 times by hand is a gentler method, and slowly turning the tube on a motorized rotisserie-style mixer for 30 min is gentle enough for DNA of any length. If necessary, try each method on a test sample and check for shearing on a gel.
  4. Centrifuge at top speed (i.e. 12,000-14,000 rcf in a microcentrifuge) for 1–2 min to separate the phases.
    • If the interface is disturbed, try letting the rotor decelerate without braking.
  5. Remove the aqueous phase to a new tube.
    • The aqueous phase is usually the upper one. However, the difference in density is minute, and salts will invert the phases. Phase inversions are usually obvious because the organic phase is colored by the antioxidant. When the mixture is of water above 25:24:1 or phenol:chloroform, inversion is more difficult.
    • For valuable samples, the loss of DNA into the organic phase can be reduced by adding a second volume of water, mixing, centrifuging, and removing again. The second volume is combined with the first if space permits, or carried through the procedure in a separate tube. The added trouble of these extra tubes outweighs the benefits of increased yield for less valuable samples.
    • Removing the lower phase first can make the upper phase easier to remove.
  6. Extract with a volume of phenol:chloroform.
    • Phenol is used for the first extraction because it removes proteins well. However, especially after repeated extractions, the aqueous phase will take in some phenol, and this step removes it to avoid interference in procedures downstream. Using phenol:chloroform or 25:24:1 instead of phenol for the first extraction reduces the amount of contamination, but many workers will extract again with phenol:chloroform.
    • Optionally, extract once more with chloroform. This is almost never necessary, but will ensure that no phenol whatsoever remains.
  7. Ethanol-precipitate the nucleic acid

[edit] See Also

[edit] References

  1. ^ Chomczynski, P. & Sacchi, N. (1987). "Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction". Anal. Biochem. 162: 156–159. doi:10.1016/0003-2697(87)90021-2. 
  2. ^ Chomczynski, P. & Sacchi, N. (2006). "Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: Twenty-something years on". Nature Prot. 1: 581–585. doi:10.1038/nprot.2006.83. 
  3. ^ TRI reagent [1]
  4. ^ TRIzol [2]
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