phenol chloroform isoamylalcohol - When do I add the TRIS buffer? (Jul/13/2007 )

Dear All,

I've got such a simple question, but no-one seems to tell me the answer. I ordered some of this phenol chloroform isoamyl alcohol from Fisher Scientific and it came with a separate bottle of TRIS buffer.

My question is, when do I add this? Do I now add the whole tris bottle to the other one, aliquot and freeze? or do I add them as I need to.
Fisher finally told me to add 45ul to every 1ml, but this doesn't seem to get the right pH. I pH'd it with a universal indicator solution and it doesn't get to pH 7-8.

I also tried a test DNA extraction and didn't see any strands.

Can someone help me please? There is one person at Fischer who knows the answer but he never seems to be by a phone and he calls me when I am not there and leaves no helpful information. Sorry to sound bitter but fed up of trying to contact the *one* person there who *might* know the answer!!

Hi there,

The entire bottle of Tris buffer that comes with the PCI should be added before use. The pH is temperature dependant. Before extracting with PCI from fisher allow it to warm to room temp. Make sure sample is mixed until a good emulsion forms to ensure best extraction. hope this is helpful.

We use the sigma one. It comes with a bottle of TRIS buffer. The whole bottle is added then keeps the mix at 4°C up to 2 years. I think it would be the same.....

Hi,

Many thanks Mauria and Aztecan Princess for your help with this, it has been causing me a real headache and it should have been so obvious!!

Thanks again,

Newbie