I am trying to learn the basics of phenol-chloroform extraction of DNA. I use the wikipedia article for it http://en.wikipedia.org/wiki/Phenol%E2%80%93chloroform_extraction However I have a problem with a part of their explenation:
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.
Now what I dont get is this: why is there a chaotropic denaturing solution? If you add this, wouldnt you precipitate your DNA as well? I guess this is why they state: "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" ? But is this not true for RNA? DO you need a chaotripc salt for RNA?
A second question: RNA , why do they state that RNA is recovered? Isnt DNA also recovered?
Second part of their eplenation:
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
Not sure how to understand this. So they add guanidinium thiocyanate to denature the proteins and seperate the rRNAs from the proteins, but if you add this salt, will you not also precipitate the RNA? Or they use a certain concentration that only denatures the proteins and not precipitate the RNA or?
