I'm trying a genomic DNA extraction protocol that uses 4% CTAB, 3M NaCl, 2.5% PVP-40, 20mM EDTA (pH 8), 100mM Tris-HCl (pH 8) and 3% beta-mercaptoethanol as lysis buffer. I incubate at 65oC for 60 - 80 min. Then, precipitate complexed cell debris with chloroform:isoamylalcohol (24:1). Collect the upper fase and precipitate DNA with isopropanol.
I tried this procedure in 51 samples of cotton leaves. In most samples I obtain a nice DNA precipitate after adding isopropanol. However, in 12 of them, I obtain 2-phases, with no DNA precipitate at all. The lower phase looks yellowish and sometimes a bit dark. Why would this happen? Any thoughts? Thanks!