rna isolation - (Aug/28/2009 )

hi all
Im trying to do rna isolation from plant tissue but im having a little doubt regarding the use of phenol

Why do we use phenol calibrated with tris pH 8 along with chlroform and isoamylalcohol?Is it required or specific for rna isolation because for dna isolation also we use phenol of ph 8, during the isolation of this rna is thre any chance of dna contamination.


plz help me with my doubts.
thanks in advance

deespike,

I'm not entirely sure where your question lies, so let me go over the basics and I'll try to touch on everything I think you may be asking.

The phenol / chloroform will separate into different phases. The isoamyl alcohol will reduce foaming, and also reduces RNA loss at the interphase due to protein-RNA interactions (I think). Nucleic acids will separate into the upper aqueous phase. Proteins will lie mostly at the interphase.

If you're using a pH of 8, you'll get all nucleic acids in the aqueous phase. If you drop the pH to around 4 or 5, you should get only RNA in your aqueous phase, as the DNA will become insoluble around pH 6. If you keep the pH at 8, you'll have to precipitate out the RNA afterward which you can do fairly easily with ethanol.

Cheers,
-Carlton