Phenol:Chloroform:Isoamyl Alcohol preparation - (Jun/12/2007 )
I have a (stupid) question: How to prepare a solution of Phénol:Chloroform:IsoamylAlcohol (25:24:1)??
I've found multiple protocols on the web (with Tris or without...) so I'm lost!!
please Help me
you don't need a protocol...
The 25:24:1 means that you take 25 parts (e.g. 25ml) of the first (Phenol), 24 parts of the second (Chloroform) and 1 part of the last (Isoamlyalcohol) to get your mixture.
the Tris acétate or anything else... is to buffer the phenol.
Mixture of PCI should be prepared with 25 volumes of phenol 24 volumes of cloroform and 1 volume of isoamylalcohol.
In general, chlorofom and IAA are premixed for convenient use. So it's an equal mixing of both solutions.
The other point is the pH of the phenol you use.
For 8, you separate both DNA and RNA and at 4.5 - 5 it's only RNA.
Normally I try to make all buffers and solutions by myself, as also Phenol:chloroform:isoamylacohol. But I want to advice you not to make this solution by yourself. First you have to melt the phenol which is available in crystalline form. Then you have to produce tris-buffered phenol (for DNA extractions) and that is very time consuming. Additionally, It is not so simple to check the pH in Phenolic solutions (pH strips could not be used, for pH meter measurements you have to mix the phenol with methanol as far i can rememver).Then you have to by 8-hydroxyquinolin, which is an antioxidant used to conserve the phenol.
And one time I want to use it, it shows a yellow discoloration. When it turns yellow it should not be used anymore.
After that i decided to buy me some Tris-Buffered Phenol and Phenol:chloroform:isoamylalcohol solutions:.
That was my exeprience with making Phenol:chloroform:isoamylalcohol solution, but don´t keep you from making your own solution.
we make phenol chloroform soln by ourself. yes it takes atleast 3 days to reach the desired pH 8.
we use tris to buffer the soln. we check pH by pH stirp. in about 3-4 months the solution truns to yellow and we make new one to use