Phenol+pH for RNA extraction - phenol-chloroform isoamylalcohol extraction (Aug/19/2005 )

Hi there,

This might be a really stupid question but I am going to start working with RNA and want to perform a phenol-chloroform-isoamylaclohol extraction.

For RNA work, the phenol should have a pH of 4.7 - right? We just have Tris buffered phenol - can I adjust the pH? If so, how???

Also, when setting up the PCIA soultion, I simply add 25:24:1 volumes of each, wrapp it in foil and store it at 4°C - is that right?

I am really kind of desparate at the moment . Thanks a lot for any advise!

Freiberger

hi
in ordr to check pH of phenol solution, ambion's ebsite gives adds here. For RA extraction, pH should be 4.7.

Prepare a mix of PChlo IAA, at the indicated proportions (you are right of them) mix vigourous and add 1cm of Tris buffer in order to decrease the evaporation.

Fred

Hi, i use invitrogens Trizol, i put some zirconium beads in the bottom of a 1.5ml eppendorf, upto the 0.5ml mark and then i used a pelleted 50ml cell culture which was washed twice in UPW and resuspended in 0.750ml trizol, i bead beat for 30seconds, then leave for 5minutes and add the chloroform, it is practically a no fail RNA extraction which is quick and simple, if interested please contact me and ill send you my protocol.

Thanks Microman,

I just got the Invitrogen Trizol too. But that wasn't actually my question ;-). However, I solved my problem.

Freiberger

For long term storage (more than a few months) you should really store the PIA at -20C or lower. This will help prevent the phenol from oxidising.

Daniel

Sequencing DNA programs