3 replies to this topic

[k_undertoe]

So, I've seen several recommendations when doing phenol:chloroform extraction to first use a saturated phenol:chloroform:isoamyl alcohol solution followed by a second extraction using only chloroform.  I have the correct buffered and saturated PCI solution, but now I am at a loss: what exactly constitutes a 'tris-saturated chloroform' and more importantly how do I make it?  I have now some pure chloroform (well, alcohol added as a stabilizer...) but what do I need to do to it in order to use it in my extraction?  The specific product I have is this: spectrophotometric grade ACS reagent chloroform, Sigma 366919(C-5312)

Edited by k_undertoe, 30 October 2012 - 08:44 AM.

[hobglobin]

Actually I never did it but the recommendations from protocols (and many bioforum posts) all have the same content: buy it as ready-made mix as this doing it yourself is a bothersome and not that easy process. And the finished stuff is cheaper too.

[phage434]

Chloroform has no acid/base groups, and is largely insoluble in water.  You can use chloroform without further treatment or pH control, unlike phenol-chloroform or phenol.

[memari]

I have never heared that until now.
Maybe you mean saturating phenol with Tris (pH 7.9 for DNA and pH 5 for RNA)

https://www.google.c...iw=1280&bih=835

Edited by memari, 03 November 2012 - 08:25 PM.