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Chloroform/Isoamyl Alcohol extraction. Problem with phases separation. - (Nov/23/2011 )

I'm working on this protocol to isolate RNA from a fern, and there's a step of centrifugation with a mixture of phenol(pH5)/chloroform/isoamyl alcohol (25/24/1), followed by a step of centrifugation of the upper aqueous phase mixed with a mixture of chloroform/isoamyl alcohol (24/1). After the 1st centrifugation I got great separation of phases and was pretty easy to collect the aqueous one; but I couldn't see any separation after the 2nd centrifugation. Any idea why this could happen? I'm trying again tomorrow, but since it's a 2-day protocol, I want to be sure it won't happen again so I won't lose another 2 days.

Thanks for your help!


Are you sure you collected the aqueous phase in the first spin? The phenol/chloroform phase will mix with chloroform and not give a phase separation. The aqueous phase should be the upper phase.


i agree with phage.... and if you separating aqueous and chloroform them you will see two transparent layers, and the upper one is aqueous...if you remove the aqueous from chloroform mixture and spin again for second time sure the chloroform is not much as the first spin, unless you mix again with chloroform before you proceed to 2nd spin...i guess you do it right just you are rushing about time...keep cool and keep doing well...:) _Evanescence


I did collect the upper phase after the 1st spin, don't know what happened then, let you know when I try again.


For me if you took 1st layer and do spin one more time, sure you wont get 2 layers...why? because the phenol and chloroform was bind each other..remember the function of chloroform is to remove if you cant get the two layers after the second spin the for me it;'s okay...but sometimes people do add some amount of chloroform after 1st spin ( collected aqueous+ chloroform) for 2nd spin just to ensure no remain phenol left, and if you do this then you will see two layers, if you not doing this then you wont see it...Good luck!


If I understand it right, Flopilus writes he mixed the upper phase with the chlorophorm for the second, but didn't get two layers.
This may be the case of what phage434 wrote, I have encountered once or twice or so, that after centrifugation with phenol, the layers were reversed (we know that, because our phenol is yellow), phenol in the upper phase.

So for first isolations, I would collect the upper phase from first centrifugation but keep the other one and try to smell them to tell which one is aqueous. Then add chlorophorm to the expected aqueous, the layers should be formed immediatelly. If they don't, try to add the chlorophorm to the phase left by and see what it does.