2 replies to this topic

[claritylight]

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?

[pDNA]

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.

Regards,
p

[David C H]

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.