The upper aqueous phase appears in the bottom of the tube.
After adding chloroform you must mix the sample properly by turning the microcentrifuge tube up and down. Then incubate it for about 5 minutes before centrifuging.
After adding chloroform you must mix the sample properly by turning the microcentrifuge tube up and down. Then incubate it for about 5 minutes before centrifuging.
Hi,
Did you see this occurring immediately after you added the chloroform? If so, Prerana is correct - you must mix the Trizol-Chloroform thoroughly (15 seconds of inversions should be sufficient). Your Trizol will now go cloudy. Centrifuge for 15-30 mins, 10,000g and you'll get separation.
Following centrifugation, you should not see the clear aqueous phase in the bottom of the tube. You should have the aqueous phase on the top, a white opaque intermediate phase (which differs in size depending on the amount of starting material) followed by the phenol layer. I have placed our tried and tested RNA extraction protocol below:
1. Add 1ml TRIzol to each well of a 6-well plate.
2. Allow the cells to lyse (
Thank you. I followed the trizol protocol, but it didn't work out. It worked when I added 100 ul of chloroform and mixed and centrifuged again.
I also faced the same problem.A gentle invert mix just after adding chloroform followed my 2-3 minutes incubation at room temperature before centrifugation gives a better result.
Same issue here. The protocol didn't mention mixing after adding chloroform (we use 1-Bromo-3-chloropropane), so the student didn't do it and we got the aqueous phase on the bottom. What a difference a few seconds of inverting makes!
If somebody has this issue, maybe this could help: I had today this problem, despite I mixed very well my samples and I did exactly the same thing as before (and it worked). Probably either trizol or chloroform went bad. So I added again freshly opened trizol/chloroform (5/1 ratio) to my samples which are already in the "bad" mixture, mixed well, and it solved the issue, pink again in the bottom and aquaous phase in the top.
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