Talk:Acid guanidinium thiocyanate-phenol-chloroform extraction

Comment
I am not a fan of wiki's policy on how-tos but this is against the rules, so I copied it here before someone deletes it. read it here (hidden: go to edit or raw mode to retrieve) <!--

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.
 * 2) * 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.
 * 3) * 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.
 * 4) Add an equal volume of phenol to the tube.
 * 5) * Phenol:chloroform:isoamyl alcohol will give a sharper interface, and 25:24:1 will show less foam.
 * 6) * 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.
 * 7) Vortex vigorously to mix the phases.
 * 8) * 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.
 * 9) Centrifuge at top speed for 1–2 min to separate the phases.
 * 10) * If the interface is disturbed, try letting the rotor decelerate without braking.
 * 11) Remove the aqueous phase to a new tube.
 * 12) * 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.
 * 13) * 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.
 * 14) * Removing the lower phase first can make the upper phase easier to remove.
 * 15) Extract with a volume of phenol:chloroform.
 * 16) * 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.
 * 17) * Optionally, extract once more with chloroform. This is almost never necessary, but will ensure that no phenol whatsoever remains.
 * 18)  Ethanol-precipitate the nucleic acid -->

About the merge I am not sure about the direction --Squidonius (talk) 13:49 & 13:53, 18 April 2008 --Squidonius (talk) 14:28, 22 April 2008 (UTC)(UTC)

Merged as most editors here are theory people and not methods people. --Squidonius (talk) 23:54, 7 May 2008 (UTC)

guanidinium-cl/phenol/chloroform extraction too  ....but basic phenol/chloroform-extraction as commonly referred to  wouldn't include the guanidine as well!! ** — Preceding unsigned comment added by 62.6.248.170 (talk) 11:50, 10 November 2011 (UTC)
 * This article is wrong on several fronts. It's entitled phenol-chloroform_extraction,  therefore should present that alone (or have separate section(s) or articles if necessary)  for phenol/chloroform extraction,  acid-phenol/chloroform extraction, choroform/iaa washing,  salt/ethanol(or IPA) precipitation  etc....   and yes include

This article totally ignores the use of PCIA extraction for DNA purification, where it is frequently indispensable for final preparation for transfection or other procedures sensitive to protein contamination. Also some parts of the article are not consistent with what I has been explained to me about the way the procedure works (eg. I've been told that proteins will crash out at the interphase due to disruption of phenolic rings in the protein, not dissolved in the organic layer). I'll verify some of this stuff and I will probably modify the article to some extent. Vladdybhs (talk) 15:41, 6 August 2008 (UTC)
 * I mostly agree: the 'How it works' setcion describes procedure for RNA, and is not valid for DNA. At least, the subsection title should be ammended. --AngelHerraez (talk) 11:58, 3 March 2011 (UTC)

I changed: "... and separates rRNA from ribosomes" to "... and separates rRNA from ribosomal proteins".

Furthermore: "... while phenol, isopropanol and water are solvents with poor solubility" --> poor solubility for what? This sentence is unclear.

--Felix Tritschler (talk) 17:17, 8 October 2012 (UTC)

Are you sure about the role of guanidine salts in phenol-based nucleic acid-extraction? This contradicts most protocols for phenol-extraction. Most of these protocols do not use guaninide salts at all. Phenol itself is already a chaotrophic agent. Also, according to most extraction protocols, the separation of RNA and DNA is dependent on the pH of the phenol solution (not any additional chaotrophic salts). At low pH mostly RNA is found in the aqueous phase, at high pH both RNA and DNA are found in the aqueous phase.134.76.70.252 (talk) 16:39, 4 February 2013 (UTC)

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