phenol-chloroform extraction - (May/28/2013 )

Hallo all,

Is it possible there is "a mistake" on the wikipedia page that describes the phenol-chloroform extraction of DNA ? (http://en.wikipedia.org/wiki/Phenol%E2%80%93chloroform_extraction)

I am especially refering to this:

This method relies on phase separation by centrifugation of a mix of the aqueous sample and a solution containing water-saturated phenol, chloroform and a chaotropic denaturing solution (guanidinium thiocyanate) resulting in an upper aqueous phase and a lower organic phase (mainly phenol). Nucleic acid (RNA, DNA) partitions in the aqueous phase, while protein partitions in organic phase. In a last step, RNA is recovered from the aqueous phase by precipitation with 2-propanol or ethanol. DNA will be located in the aqueous phase in the absence of guanidinium thiocyanate and thus the technique can be used for DNA purification alone.

Guanidinium thiocyanate denatures proteins, including RNases, and separates rRNA from ribosomal proteins, while phenol, isopropanol and water are solvents with poor solubility. In the presence of chloroform or BCP (bromochloropropane), these solvents separate entirely into two phases that are recognized by their color: a clear, upper aqueous phase (containing the nucleic acids) and a bright pink lower phase (containing the proteins dissolved in phenol and the lipids dissolved in chloroform). Other denaturing chemicals such as 2-mercaptoethanol and sarcosine may also be used. The major downside is that phenol and chloroform are both hazardous and inconvenient materials, and the extraction is often laborious, so in recent years many companies now offer alternative ways to isolate DNA


When you want to extract genomic DNA: you do not add the chaotropic salt? RIght? If you do, you would also precipitate the DNA (and thus the DNA would not be in the aquous phase anymore...

I think its a bit confusing (not so much a mistake).

Or ?

But if you use that salt to seperate the rRNA from ribosomal proteins, how do you make sure the salts will not precipitate the RNA too (like it does with the proteins) since I have always been told that salt will also precipitate DNA.

ALso: the state RNA is recovered from the aqueous phase, but what with DNA? This is recorved too..?

They are confused. I'd edit it to fix it, but I've sworn off editing Wikipedia after being insulted multiple times by their "editors."

Read a real protocol. Openwetware, for all its faults, is better than this.

phage434 on Wed May 29 20:37:09 2013 said: