dna extraction question - (May/09/2011 )
What is the difference in using phenol:chloroform:isoamyl alcohol at 25:24:1 v/v versus just using chloroform:isoamyl alcohol at 24:1 v/v?
I need to extract some DNA but seems there is only the phenol:chloroform:isoamyl alcohol left but the protocol just calls for chloroform:isoamyl alcohol so I am wondering what the difference would be?
Chloroform helps to denature proteins as well as removing residual phenol (since phenol is not very soluble in chloroform) ...so it is not a really good idea to use phenol:chloroform instead.
You can try to find some chloroform without isoamylalcohol ...that would do the trick as well. The isoamylalcohol is not that essential.
In addition to denaturing protein, phenol also acts as a buffer and is used in RNA isolations to keep the pH acidic. Acidic phenol:chloroform is used for RNA isolation; at this pH DNA will be seperated into the interphase and RNA will be left in the aqueous phase. If the pH of your phenol:chloroform is below 7, you will lose DNA. I believe the isoamyl alcohol acts as a defoaming agent, probably allows the phases to mix more completely and efficiently.