inverted phenol and water phase during DNA isolation? - (Jun/04/2007 )
I was doing DNA extraction from skin tissue from 20 different samples. So I treated them with proteinase K and did a phenol-chlorophorm-isoamyl alcohol extraction as I always do. But in two of my samples, after adding phenol and centrifugation, I got phenol phase (th yellow one) in the UPPER part of the tube, and the transparent water phase in the bottom (they were inverted!! ). All the other samples behaved normally. Did it ever happen to you? Can you do DNA isolation from the bottom phase? Has anyone tried?
may happen if it's not well homogeneized and if spin is done at 4°C.
vortex and spin at RT should be fine
or is there any chance you take the upper part of phenol-chloroform for that two sample???
This can easily happen with high salt in the water. The density of phenol and water is quite similar, and the salt can push the water density higher. Phenol chloroform does not have this issue.
Yes, I've seen it with high salt extraction buffers. But that was with straight phenol, not phenol+chloroform. Chloroform is pretty dense so it shouldn't happen...
thank you all
anyway, just so you know, I tried to extract DNA from the water phase that was in the bottom part. It worked just fine, and I got a decent concentration. So, it works even if the phases are inverted.