DNA extraction - why 100% and 70% ethanol? (Apr/02/2010 )
thanks a lot phage434
josse on Apr 16 2010, 11:10 PM said:
Why do people add Sodium acetate when doing an ethanolprecipitation?
If you look at the details of the old Birnboim and Doly method (aka alkaline lysis) the gDNA and proteins are precipitated with potassium Ac, and there is also a good protocol using ammonium Ac for DNA precipitation, which is good for smaller DNA fragments. I think Na is often used because you need less to precipitate the DNA.
ah interesting swanny,
I'll check that
ok, now I am a bit confused,
lets say we use salt precipitating method, or commercial kits such as promega wizard genomic DNA purification kit, the common step is using a cell lysis solution (contains ~2% SDS, EDTA Tris), Protein precipitation solution (~7.5M ammonium acetate)...
As was discussed, the sodium or ammmonium ions is to coalesce and precipitate DNA, but when ammonium acetate is added to precipitate proteins, will it be precipitate out the DNA as well? This is because after the ammonium acetate being added, we incubate the sample on ice, then centrifuge down the pellet (which I believe is protein pellet), and transfer the supernatant for DNA precipitation using alcohol. IF the DNA had been precipitated when ammonium acetate being added, the supernatant we transfer shouldn't contain any DNA.... so how is it actually happen?