having a rotten experience with my phenol.... - (Mar/16/2006 )
I have a really dumb problem and I am hoping someone can help me, pretty please?
In the past I have used phenol to extract nucleic acids. It has always been pre-equilibrated when it was bought. Well, I opened a new bottle that needed to be equilibrated before use.
I got the protocol from Maniatis. You are supposed to stir the daylights out of the phenol in a beaker/flask with a bunch of tris...pull off the tris...repeat until the phenol is the right pH
well, I spent about 5 hours stirring tris into that *%@# phenol yesterday and the pH would never come out properly (I made up fresh tris buffer and checked it on two pH meters to make sure that wasn't the problem). I changed the Tris about 20 times and used a much higher volume than what was called for, when I realized it wasn't going to work easily
Is there a secret trick to getting this to work? I attempted my extraction with the pH off anyways because I need some good clean DNA, and of course it didn't work (I am prepping genomic DNA and so can't use my handy little spin columns; they are for little PCR products). I ordered some equilibrated phenol so I won't have to go through this again, but it is on backorder till late next week and I could really use the DNA yesterday....
Does anyone know a trick to this? Am I missing something silly that's making it not work?
Thank you oodles to anyone who can help me solve this!
well. Can be a stupid proposal, but to check the pH of a saturated phenol, you need to solubilize the phenol in an aqueous medium. The following method is used by ambion to check pH...
*For phenol:chloroform:IAA or acid phenol:chloroform solutions - mix 2 ml of the organic phase with 8 ml of methanol and 10 ml of water. Measure the pH of the entire sample.
*For saturated phenols - mix 2 ml of the organic phase with 5 ml of methanol and 13 ml of water. Measure the pH of the entire sample.
(here) is the link of ambion's web page to that point.
It may not be feasible, but as an alternative I would suggest a salting out protocol in place of phenol/chloroform? PM me for a good protocol of you need one aimikins
thank you for the advice, Fred; I will try it that way.
I was flooding pH paper with the phenol using a pasteur pipet (I was concerned that it would damage the electrode) so perhaps I was not getting an accurate reading. I will try it again.
John, I do think I will send you a pm for that protocol; I always like to have alternatives on hand
thank you, gentlemen!