Phenol ChCl3--upper or lower phase (?) - (Oct/22/2012 )
Hello: I recently purchased a bottle of acid phenol/Chloform/IAA from Ambion, and on standing it separates into two phases--most of the volume is in the lower (denser) phase.
I wanted to use this for an RNA extraction. Do I:
1.) Swirl the bottle well before I use to mix the phases well, and then take out what I need, or
2.) Let the bottle stand, and take out solely from the lower phase?
Thanks for any consideration.
Sounds as if the mix is equilibrated with buffer which makes the upper phase, so follow the 2nd idea....
I thought that the upper phase was some kind of buffer as well. However, I looked at the MSDS and it said that there was phenol, ChCl3, and IAA. It did not mention anything else, like Tris. So I was suspicious the upper phase was something useful to the extraction.
actually I'm not sure with RNA extraction mixes...better wait for another comment here...I'm only used to ph 7.9 phenol/chloroform for DNA...yours surely has pH 4.9....
Lower phase only. The upper phase is buffer for controlling the pH of the phenol solution. For RNA work, the pH should be around 4.5.
Definitely the lower phase. That top layer is just a buffer to keep it acidic. It's equilibrated that way intentionally. If you mix it up, you will lose some of the properties of the phenol.