I recently performed a DNA extraction for a microbiome project from cotton rectal swabs and urine pellets using phenol-chloroform. Briefly:
1. Swabs were incubated with a mutanolysin/lysozyme enzyme cocktail for 1h @ 37C
2. Added glass beads, phenol:chloroform:isoamyl alcohol (25:24:1) and 20% SDS, then agitated (TissueLyzer/bead beater) and centrifuged @ 14k rpm x5 min.
3. Aqueous layer transferred to a clean tube, and added 3M sodium acetate and ice-cold isopropranol at -20C overnight.
The next day, no pellet! I pulled off the supernatant carefully (assuming there might be a pellet I couldn't see) and re-constituted in water, ran a PCR and a 1% agarose gel but no DNA bands! I ran the sample on the UV-Vis (Nanodrop) and got nothing (no peaks, no DNA).
I also tried UV-Vis on the supernatant and got what looks like a phenol peak at 270 and also a second peak at 260, which I think is DNA (see attached).
My interpretation is that the aqueous supernatant (with NAOAc and isopropanol) still has both phenol and DNA in it, the former being a technical error I suspect. I've tried adding more NAOAc or isopropanol or even repeating the phenol-chlor extraction with aliquots of this supernatant, but to no avail.
Does anyone have suggestions on how to pull the DNA out of this solution?