Phenol - why different for DNA and RNA extraction? (Jun/20/2009 )
Why is phenol of alkaline pH is used during DNA extraction and that of acidic pH during RNA extraction? What is the exact molecular basis for this difference? Phenol itself has got acidic pH. Why then is it "neutralized" for RNA isolation? How to equilibriate phenol with water for RNA extraction?
What do you think? Check out the Protocols pages for equilibration of phenol.
bob1 on Jun 21 2009, 06:11 PM said:
Thanks for the reply Phage
I could find answer of my first 2 question there, but not of the 3rd question. Why to saturate phenol with water for RNA extraction? What would happen if directly pure phenol is used?
Unsaturated phenol, used to extract an aqueous sample, would become saturated -- with the water in which your RNA is dissolved...
HomeBrew on Jun 24 2009, 06:59 PM said:
So what? Let it saturate! Generally equal volume of phenol is used. So not all water would go into phenol. If the purpose of removing proteins and DNA from RNA is served then pure phenol also should be fine! Has anybody ever used pure phenol for RNA extraction??
Pure phenol is a solid, and difficult to manipulate. It might work, by why would you want to do this?
Is that the only reason?
I am interested in it because it would avoid the major step of "preparation of saturated phenol"
Dissolving solid phenol in excess water does not seem like a major step to me. You can also purchase it as a solution, which I would recommend, especially for the higher pH version equilibrated with Tris at pH 7.5.
We generally do the saturation 3 times with water. When I did this last time, the phases did not separate even after keeping for 3-4 hrs. I had to keep it overnight at 4 C to separate the phases. So it took total 3 days. Do you mean saturation just once is enough to keep the phenol in liquid state at RT (if that is the only purpose)?