I'm looking for help identifying why ~40% of the time I do not see a pellet during my RNA extraction (TRIZol, not a spin kit). Instead I get another phase separation (see photo). Other times the pellet shows up fine, with no deviations from the protocol between experiments. My protocol involves two additions of chloroform, then isopropanol with glycogen.
I have tried vortexing the tube and spinning again, and this sometimes fixes the problem but not all the time. Any help identifying the mystery layer at the bottom is appreciated!
I have exactly the same problem and no one could answer it. Please if you find out why this happens can you inform me too?
Hi, after the addition of chloroform, you should indeed have 3 layers (the lower is the remaining pheno-chloro phase, and you have an interphase). Your RNA should be in the upper phase (the aqueous one). By tilting slightly the tube, pipette carefully the upper phase into a fresh tube. After this, you can proceed with precipating your RNA from the new tube by adding cold isopropanol and glycogen. The RNA pellet usually is very small and appears (as a very small spot) after centrifuge at 4 degrees. Hope that helps a bit..
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