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Trizol contamination problem with Drosophila RNA Prep - (Aug/16/2007 )

Hi,
I'm doing RNA preps using the trizol extraction method from Drosophila. Following isopropanol precipitation, i get a very small drop of pink/orange organic phase following centrifugation. This pink pellet is also where most of the RNA is prescipitated so it cannot be removed. I definitely didn't carry over any of the organic phase following chloroform - trizol extraction and i did the chloroform extraction twice, and i'm using new reagents.

The droplet only appears after addition of isopropanol. And it prevents the pellet from drying after ethanol wash. If i go to the end of the method, the RNA is good quality (i.e. 260/280=~2) but there is contamination (260/230 low i.e.<1).

It appears as though some of the organic phase is staying in solution in the aqueous phase as it has a distinct yellowey/orangey colour to it following chlorofoem extraction. It then comes out of solution with addition of isopropanol.

Can anybody shed any light on why this is happening please and what i can do to get a clean pellet. Thanks in advance for your advice.

Tim

-tim s-

may you try a new bottle of trizol? the phenol of the trizol may be oxidized, but you can't see it as the trizol is so pink. If your 260//280 ratio is good, means you don't have proteins. So do an other chlorofom only step (removes phenol and more hydrohpobic).
Alternative, add 1ml butanol on your "orange" pellet. All RNA will be pelleted, and phenol is butanol soluble.

-fred_33-

QUOTE (fred_33 @ Aug 16 2007, 11:54 PM)
may you try a new bottle of trizol? the phenol of the trizol may be oxidized, but you can't see it as the trizol is so pink. If your 260//280 ratio is good, means you don't have proteins. So do an other chlorofom only step (removes phenol and more hydrohpobic).
Alternative, add 1ml butanol on your "orange" pellet. All RNA will be pelleted, and phenol is butanol soluble.


Thanks for the advice. However, it doesn't seem to have helped. I'm using a new bottle of trizol and i've already done an extra chloroform phenol extraction step (so two extra now). I tried adding butanol to precipitate the RNA instead of isopropanol but the orange droplet still appears following centrifugation. This suggests that it's not phenol as it doesn't dissolve. Could this be something specific to drosophila or has anyone else encountered this problem. I can't understand it as it even recommends a trizol extraction on the Flybase website.

Tim

-tim s-

QUOTE (tim s @ Aug 17 2007, 01:58 AM)
QUOTE (fred_33 @ Aug 16 2007, 11:54 PM)
may you try a new bottle of trizol? the phenol of the trizol may be oxidized, but you can't see it as the trizol is so pink. If your 260//280 ratio is good, means you don't have proteins. So do an other chlorofom only step (removes phenol and more hydrohpobic).
Alternative, add 1ml butanol on your "orange" pellet. All RNA will be pelleted, and phenol is butanol soluble.


Thanks for the advice. However, it doesn't seem to have helped. I'm using a new bottle of trizol and i've already done an extra chloroform phenol extraction step (so two extra now). I tried adding butanol to precipitate the RNA instead of isopropanol but the orange droplet still appears following centrifugation. This suggests that it's not phenol as it doesn't dissolve. Could this be something specific to drosophila or has anyone else encountered this problem. I can't understand it as it even recommends a trizol extraction on the Flybase website.

Tim

try to wash with 70% ethanol twice...260/230 ratio will increase significantly...at least it did in my case...macrophage RNA isolation using tri reagent..
good luck

-yasd-