Buffered Phenol Troubleshooting - (Oct/20/2006 )
Hi,
We make buffered phenol (saturated phenol) for RNA extraction buffer by mixing several times with a Tris-HCl solution. Normally, the color of the phenol does not change that much, but this time we are seeing that the solution turns a very dark brown (almost black) color when the phenol and tris are mixed. Has anyone experienced something like this before, or can you tell me what might be happening? Has the liquified phenol gone bad?
Thanks for your advice!
Jenn
Hi,
Did you add hydroxyquinoline (0.1%) and phenol together? Seems to be like phenol oxidation, but can't be certain. Could also be dued to contamination.
Did you add hydroxyquinoline (0.1%) and phenol together? Seems to be like phenol oxidation, but can't be certain. Could also be dued to contamination.
No, we did not add hydroxyquinoline. Our recipe for buffered phenol requires you to mix an equal amount of unbuffered Tris with the liquified phenol for 10 minutes. Then you decant the top layer and repeat this with Tris pH=8.0 for several steps. In the end you store it mixed with 0.5 the volume of Tris pH=8.0. Normally it works fine...I'm not sure why this happened. Maybe a bad batch of phenol?
Hi,
Seems like oxidation. Refer to this link for more info http://plantpath.unl.edu/llane/text/phenol.html.
Don't really know if it's a bad batch of phenol. If it is, you could exchange it for another new batch, I guess.
Bests
Hi,
normally phenol turns pink if oxidation occurs but any way you shouldn't use it. It really could be a contamination. But I am just wondering why did you buffer saturate it at all?
Wou wrote you want to isolate RNA. RNA needs water saturated phenol pH 4.0-5.0 to stay in aqueous phase. You took Tris-HCl Buffer, making the phenol basic. If pH is bigger than 7, RNA is bound to the organic phase and only DNA remains in aqeous.