Ratio of phenol:chloroform in RNA extraction - 1:1 or 5:1? (Jun/01/2006 )

Hi all,

I was just wondering what is the optimal ratio for phenol:chloroform when doing RNA isolation?

The Ambion website (http://www.ambion.co.jp/techlib/tips/handling-rna.html) mentions a 1:1 ratio but Sambrook (Chapter 7 Protocol 1) seems to recommend adding 0.2ml chloroform-isoamyl alcohol to 1 ml of phenol which comes to a ratio of 5:1

Why this difference and what difference does it make to the RNA isolation?

Thanks

I've always used a 25:24:1 phenol:chloroform:isoamyl alcohol for my RNA extractions and have never had a problem. Just be sure that the phenol that you're using is acidic (pH 5.2?), otherwise you'll get horrible yields. Search the Ambion tech notes about that if you'd like more info.

_Hank

well, i use as hank a 25:24;1 phenol:chlo:IAA solution.
I remeber that a 5:1 ratio was discussed recently on that forum for plant extraction, but result obtained by the member were quite bad. pH recommended for RNA extraction is more acidic : 4.7