- Phenol-chloroform extraction
Phenol-chloroform extraction is a
liquid-liquid extractiontechnique in biochemistry. It is widely used in molecular biologyfor isolating DNA, RNAand protein. Equal volumes of a phenol:chloroform mixture and an aqueous sample are mixed, forming a biphasic mixture. This method may take longer than a column-based system such as the silica-based purification, but has higher purity Fact|date=September 2008 and the advantage of high recovery of RNA: an RNA column is typically unsuitable for purification of short (<200 nucleotides) RNA transcripts, such as siRNAand miRNA.
It was originally devised by Piotr Chomczynski and Nicoletta Sacchi and published in 1987 (referred to as "Guanidinium thiocyanate-phenol-chloroform extraction"). [cite journal | author = Chomczynski, P. & Sacchi, N. | title = Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction | journal =
Anal. Biochem.| volume = 162 | pages = 156–159 | year = 1987 | doi = 10.1016/0003-2697(87)90021-2] [cite journal | author = Chomczynski, P. & Sacchi, N. | title = Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: Twenty-something years on | doi = 10.1038/nprot.2006.83 | journal = Nature Prot.| volume =1 | pages = 581–585 | year = 2006]
How it works
This method is widely used and is sometimes referred to as the
TRIzolmethod, after the name of the Invitrogenproduct. [TRI reagent [http://www.mrcgene.com/tri.htm] ] It relies on phase separation by centrifugation of a mix of the aqueous sample and a solution containing water-saturated phenol, chloroformand a chaotropic denaturing solution ( guanidinium thiocyanate)resulting in an upper aqueous phase and a lower organic phase (mainly chloroform). Nearly all of the RNA is present in the aqueous phase, while DNA and protein partition in the interphase and the organic phase, respectively. In a last step, RNA is recovered from the aqueous phase by precipitation with 2-propanolor ethanol.
The name of the protocol stems from the chemicals used:
Guanidinium thiocyanatedenatures proteins, including RNases, and separates rRNA from ribosomes; phenol, isopropanoland water are solvents with poor miscibility. In the presence of chloroformor BCP (bromochloropropane), these solvents separate entirely into two phases that are recognized by their color: a clear, upper aqueous phase and bright pink lower phase.Other denaturing chemicals such as 2-mercaptoethanoland sarcosinemay also be used. The major downside is that Phenoland chloroformare both hazardous and inconvenient materials, and the extraction is often more laborious, so in recent years many companies now offer alternative ways to isolate DNA.
* "Phenol": The phenol used for
biochemistrycomes 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 alcoholsolution (sometimes called "25:24:1"). Phenol is naturally somewhat water-soluble, and gives a fuzzy interface, which is sharpened by the presence of chloroform, and the isoamyl alcohol reduces foaming. Most solutions also have an antioxidant, as oxidized phenol damages the nucleic acids.
* "Chloroform": Chloroform is stabilized with small quantities of
amyleneor ethanol, because exposure of pure chloroform to oxygen and ultraviolet light produces phosgenegas. Some chloroform solutions come as pre-made a 96% chloroform, 4% isoamyl alcohol mixtures that can be mixed with an equal volume of phenol to obtain the 25:24:1 solution.
* "Isoamyl alcohol": Some protocols include isoamyl alcohol as a stabilizing agent, while others do not require it at all.
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.
# 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.
# 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.
# 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.
# 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.
# 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.
# 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.
# Ethanol-precipitate the nucleic acid
Column-based nucleic acid purification
Nucleic acid methods
DNA separation by silica adsorption
* [http://tools.invitrogen.com/content/sfs/manuals/10296010%20pps%20Trizol%20LS%20Reagent%20061207.pdf] ]
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