I am using a ctab protocol to isolate DNA from cellulose degrading bacteria. After the steps of proteinase K, SDS and CTAB additions and the respective incubation and centrifugal periods, i carried out the step of Phenol Chloroform Isoamyl Alcohol (25:24:1) addition and centrifugation which should technically yield a clear aqueous phase that is transferred to a fresh tube to proceed with. My problem is that few of my samples yielded a white cloudy residue almost floating/ on top the clear aqueous phase. Repeated centrifugation could not get rid of it either. Could i have an insight into what this could possibly be?
This is pretty normal. There are several things, which are poorly soluble in both aqueous and phenolic phases. They form the white layer in between. You should avoid collecting this stuff together with the aqueous phase, even if you have to leave behind some of the latter.
The white stuff may include lipopolysaccharides, CTAB complexes with phospholipids, and denatured genomic DNA. If the layer looks like a crust, it's likely DNA. If it's like a "mist", which easily disperses in water phase, it's rather CTAB complexes. Lipopolysaccharides form viscous jelly-like layer.
@Victor G Stepanov.
What's usually normal is to have a possible cloudy white layer in close proximity to the interphase right? Cloudy stuff present in my samples were on the uppermost region of the clear aqueous phase. I have included a sketch in the attached file. Could it just be DNA afterall?
Thanks
Ah, my bad, I misunderstood your question. If it floats on top of aqueous phase, it is more like lipids or liposaccharides. DNA usually sits at the interphase.
It could also be CTAB/SDS micells. They can be formed if you shake the tubes very too much. You might centrifuge the tubes in a cold rotor at something like 10000 x g and see if the clouding disappears.
Genomic DNA is poorly soluble in water?
I have just used this method to extract DNA from bacteria, and saw the white stuff at the interface. But to me it looked like egg white, so I interpreted it as proteins.
What about the cloudy water phase that turns clear when transferred to fresh tubes?
That's something I experienced in the last few extractions.
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