Phenol chloroform extraction DNA: protein contamination - how to remove all protein from my DNA? (Feb/06/2006 )
I am using phenol-chloroform method to extract DNA frm frozen rat heart. my protocol is as follows: use of a retsch mixer mill to break down tissue while in extraction buffer(10mM tris, 0.1mM EDTA, 0.5% SDS), followed by proteinase K digestion overnight(50C), then 1 hr of RNase treatment at 37C. Then: one phenol extraction; 1 phenol:chloroform extraction; 1 chloroform extraction followed by alcohol precipitation. My yields(for heart) are acceptable, but the spec ratios are consistently 1.7. On an agarose gel of 0.8%, I do not see one distinct band of high molecular weight, but more a area of high density, high MW(some sheared DNA?).
Originally, i was using several P/C extractions followed by chloroform. However, with this method, the aqueous layer was almost completely obliterated by white precipitate(ie protein...), and multiple P/C did little to reduce this. Thus, I started using the phenol extraction as a first step, which seemed to clear the aqueous layer faster. Was this incorrect?
I have ordered new phenol, pH 8 and new phenol:chloroform:isoamyl alcohol, pH 8.
well actually 1.7 is not that bad.
If you want to get better ratios, check pH of phenol and check colour of it. Changes means degradation and decreases separation process. Finally,protein separation is better obtained when exac 1/1 ratio (aqueous/phnolic phase) is in your tube.
Finally, it's better to loose part of sample instead getting tiny quantities of organic phase.