temperature of phenol and chloroform - Is room temperature necessary? (Feb/08/2006 )
I have a protocol for RNA extraction from conifers that has a series of chloroform and phenol:chloroform extractions, all specified to be done at 21 degrees Celsius. I am wondering if anyone knows whether and why it is important to keep these chemicals at room temperature, as opposed to 4 degrees celsius. I ended up spinning half of my samples (with phenol or chloroform) at 4 degrees, just because it was easier (our centrifuge is kept in a cold room). However, in the end these samples appeared to have degraded RNA, whereas the ones that had been spun at room temperature had intact RNA.
Also, for extracting RNA as opposed to DNA, is it important that the phenol be at an acidic pH? I used pH8 (the protocol doesn't specify), and got a fair bit of RNA, although it appears there is a bit of DNA as well.
Thanks very much
The temperature of the solutions affects the rate at which the RNA or DNA precipitate, at room temperature, DNA is more likely to precipitate as there is much more of it and it is bigger than RNA, but at 4 degrees RNA should precipitate well. We use phenol at pH 6.0 for RNA extraction, I ahve never tested the pH to see if it affects the extraction.
separations between nucleic acids and proteins occurs better at RT.
I've been told, but only saw it 1time, that 4° increases the possibility of a phase inversion.