I am performing an RNA extraction on intestinal epithelial organoids using Trizol LS. After harvesting, homogenizing, and adding chloroform, I centrifuged the samples to separate out the phases. However, there is no clear aqueous phase at the top of many of the tubes (photos attached). Instead, there is a dense, thick interphase.
I noticed that some of the tubes separated normally, and these were the ones that contained more Trizol. I thought this may indicate a salt imbalance in the other tubes, so I added more Trizol to the ones that did not fully separate. This did not fix the issue.
I had been incubating on ice, and I read that RT may be optimal for phase separation (and that cold incubation may cause phase inversion). This also did not fix the issue.
Then, I thought I may have an imbalance in my Trizol:chloroform ratio. I added chloroform to offset the extra Trizol I added. After centrifugation, I still did not have a distinct aqueous phase in most of the tubes.
Any help would be appreciated!
Thanks so much.
There is not much difference in the density of water vs phenol even if chloroform is added to the phenol so if large amounts of protein and salts enter the aqueous phase it can have a similar density to the organic layer and separation does not work well. Try using less of the crude sample for a better separation
Hello Norman,
Do you mix the sample after adding chloroform? After adding chloroform you must shake vigorously by hand for 15 seconds. Incubate the sample at room temperature for 3 minutes.
After adding Chloroform, it is important that you mix the tube. The volume of the aqueous phase is about 60% of the volume of TRIzol reagent used for homogenization.
Follow the protocol given below.
1. Add 200μl chloroform to the tube.
2. Shake well by hand for 15 seconds.
3. Incubate at room temperature for 2-3
minutes.
4. Centrifuge at 12,000xg for 15 minutes at 4 degree C.
Best.
Malcolm Nobre
I mixed vigorously after adding chloroform, and I also tried vortexing at max speed for 15 seconds. Neither seemed to do the trick.
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