phenol equilibration?

Question

reasons of using TRIS buffer and the particular molarity... why with first o.5 M and later with 0.1M??

Answer 1

TRIS buffer just happens to be a very good buffer (maintains PH well). As to the second part, you really don't need to change concentrations, you can equilibrate phenol just fine with a 0.5M TRIS only.

Answer 2

There actually is a reason; you equilibrate it with a high concentration of buffer such as Tris, then you dilute out the buffer. The reason is that when you do phenol/chloroform extraction of cell lysate to get DNA, you inevitably will get the Tris buffer (top phase) contaminating. Well, at high molarity, you will add a bit of buffer into the DNA solution. DNA precipitation requires the presence of alcohol, low temperature, and low pH (which is why you add sodium acetate). Anyway, if your DNA is highly buffered from Tris contamination, then you won't be able to change the pH and teh DNA won't precipitation. Thus, long story short, use 0.1M tris as top layer for phenol, and avoid tris contamination when you do your preps.