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Phenol ChCl3--upper or lower phase (?)


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5 replies to this topic

#1 srpres

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Posted 22 October 2012 - 12:33 PM

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.

#2 hobglobin

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Posted 22 October 2012 - 12:35 PM

Sounds as if the mix is equilibrated with buffer which makes the upper phase, so follow the 2nd idea....

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#3 srpres

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Posted 22 October 2012 - 12:40 PM

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.

#4 hobglobin

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Posted 22 October 2012 - 12:52 PM

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....

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#5 phage434

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Posted 22 October 2012 - 02:48 PM

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.

#6 Ahrenhase

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Posted 07 November 2012 - 04:25 PM

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.




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