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Phenol/chloroform/isoamyl alcohol extraction - I think I took the wrong phase

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#1 MicroAvenger



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Posted 24 June 2013 - 03:38 PM

Hi everybody,

I am doing phenol/chloroform/isoamyl alcohol (25:24:1) extractions. The protocol I am using states the top layer is the aqueous layer, however, layer inversion could happen and if it does, the organic layer can be determined by its yellow color (though I have read comments that layer inversion will not happen with PCI).

The first prep I did I took the top layer without looking at color. When I spec'ed the preps using a program specifically for double stranded DNA I was getting 260/280s of 1.6-1.8 and 180 ng/ul dsDNA. The next prep, I looked at the layers and the top layer had a yellowish color (the bottom layer was clear), so I took the bottom layer. My 260/280s for these preps are 1.3-1.4 and 260-370 ng/ul dsDNA.

I took the wrong layer didn't I? If so, why would the top aqueous layer be yellowish and the bottom organic layer be clear?

Thank you!

#2 phage434



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Posted 24 June 2013 - 04:59 PM

Yes, you took the wrong layer. PCI always is the lower layer.

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