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