[Kikko]

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 explenation:

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

[Yggdrasil]

Whether DNA partitions into the aqueous or organic phase during a phenol-chloroform extraction depends on the pH of the solution.  Under acidic conditions, DNA will partition into the organic phase, so your aqueous phase will contain only RNA.  At neutral pH, both DNA and RNA will partition into the aqueous phase.  See http://tools.invitrogen.com/content/sfs/manuals/cms_071818.pdf

The guanidinium thiocyanate is not there to affect the partitioning of the DNA or RNA, so the wikipedia explanation is wrong.  It's mainly there to denature proteins and dissociate them from any nucleic acids that they might bind.  Also, guanidinium will help to unfold any RNases that are present in the sample.  Guanidinium thiocyanate is mainly used when preparing RNA from tissue samples (where you have a lot of proteins and RNases present), but it is not required for a phenol-chloroform extraction.  I've certainly done plenty of successful extractions without guanidinium thiocyanate present.

[Yggdrasil]

I edited the wikipedia page (https://en.wikipedia.org/wiki/Phenol%E2%80%93chloroform_extraction) to correct the explanations.  Let me know if something is still unclear.

[Kikko]

Whether DNA partitions into the aqueous or organic phase during a phenol-chloroform extraction depends on the pH of the solution.  Under acidic conditions, DNA will partition into the organic phase, so your aqueous phase will contain only RNA.  At neutral pH, both DNA and RNA will partition into the aqueous phase.  See http://tools.invitrogen.com/content/sfs/manuals/cms_071818.pdf

The guanidinium thiocyanate is not there to affect the partitioning of the DNA or RNA, so the wikipedia explanation is wrong.  It's mainly there to denature proteins and dissociate them from any nucleic acids that they might bind.  Also, guanidinium will help to unfold any RNases that are present in the sample.  Guanidinium thiocyanate is mainly used when preparing RNA from tissue samples (where you have a lot of proteins and RNases present), but it is not required for a phenol-chloroform extraction.  I've certainly done plenty of successful extractions without guanidinium thiocyanate present.

Ok thanks.
I got confused because of the weird wikipedia explenation.
I also never used salts when I did a DNA extraction with phenol-chloroform, so it got me confused as why you would need those salts. Especially since I learned they used them to precipitate the DNA in ethanol when doing a DNA precipitation.

Just one more question: the fact that the DNA will be in the phenol (organic phase) at acidic pH is simply due to the fact that the phopsphate backbone is not negative anymore due to the H+ of the adics , right? So its not polar enough more to be in the aqueous phase?

Or is there another reason?

[Yggdrasil]

I'm not entirely sure about the mechanism for the pH dependence of DNA partitioning, but I would assume that your answer is correct.  I guess that extra OH group on the RNA makes all the difference in either changing the pKa of the phosphate group or directly affecting the solubility of the RNA in the aqueous phase.

[Kikko]

I'm not entirely sure about the mechanism for the pH dependence of DNA partitioning, but I would assume that your answer is correct.  I guess that extra OH group on the RNA makes all the difference in either changing the pKa of the phosphate group or directly affecting the solubility of the RNA in the aqueous phase.

Ok thanks for the answer.