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having a rotten experience with my phenol.... - (Mar/16/2006 )

Hey, all

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? huh.gif Am I missing something silly that's making it not work?

Thank you oodles to anyone who can help me solve this!

Aimee

-aimikins-

hi
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.

-fred_33-

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

-John Buckels-

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!

A

-aimikins-