Phenol/chloroform/isoamyl alcohol extraction - I think I took the wrong phase - (Jun/24/2013 )

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!

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