Hello all,
I am performing RNA extraction/purification with Trizol on spinal cord samples, and have had trouble getting clean samples. To help, I've decided to add a second phenol-chloroform wash, but I'm concerned that the increased portion of chloroform has flipped my aqueous and organic layers. Because both layers are clear-colored on the second wash, it's very hard to tell which is the correct layer.
I am concerned about this because I've observed that adding too little Trizol can result in flipped layers on the first wash- the pink organic layer is at the top, and the clear aqueous layer is at the bottom.
During my first wash, I shook the tube and allowed it to incubate for three minutes. When I placed it in the centrifuge, there were numerous clear bubbles within the bottom/pink/presumably organic layer.
During my second wash, I noticed similar bubbles forming in the top clear layer during the incubation step. They appeared to be settling out to rest in the bottom clear layer, which was a direct reversal of the bubbles from my first chloroform extraction. Because of this, I think my layers could be swapped on the second wash.
Do you think the formation of bubbles is a good indicator for which layer is which? Could I add a little more Trizol for the second wash, just so I still have that color indicator for the organic layer?
Claire, would it help to spike the chloroform with a lipophilic dye (e.g. Oil Red O) for better identification? Residues may be cleaned up with isoproproanol later.
I suggest that you use a Pasteur pipette and try to pipette a few of the upper layer, then transfer some drops to a small TLC plate and notice the rate of evaporation of solvent, if it's the organic phase, you will notice rapid evaporation of chlorform slovent, if it's the aqueous layer water solvent will take longer time to evaporate. I don't know if this simple method will work for you or not, but you can give it a try.
I've realized that I over-thought this.
The density of chloroform is so much higher than water that layer reversal is unlikely in a second extraction.
Reversal is much more likely in the first extraction because of a higher likelihood of excess dissolved salts in the aqueous layer, especially if the tube was not shaken/vortexed before centrifugation.
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