cloudy phase during RNA extraction with Trizol - (Mar/06/2009 )
I'm new to Trizol extraction and there are many things that I still don't know.
I usually pellet down my HeLa cells (10 to the power of 6) and resuspend them in 200 ul PBS and then add 650 ul Trizol. I vortex and leave at RT for 5 min. then I add 200 ul Chloroform, shake vigorously and then centrifuge at maximum speed for 10 min at 4 degrees.
after centrifugation, there is always a cloudy phase between the clear top phase and the Trizol phase....what is that?....do I need to collect that?....is that the cell nuclei fraction?
I experienced the best results when I lyse the cells directly in the culture plate or flask with Trizol after washing with PBS. I use 1ml for a well of a 6well plate or 2ml Trizol for a T25 flask but this may also depend on the cell type.
After centrifugation you have three phases: the red organic phase, a white interphase and the clear aqueous phase. You have to collect ONLY the clear aqueous phase for further RNA isolation.
it's interesting how the cloudy interphase doesn't pellet down at that highspeed (13000 g for 10 min at 4 degrees)
do you usually see the pellet of RNA after addition of isopropanol?
Sometimes I see it, sometimes not but nevertheless i continue the RNA prep. A big white pellet indicates gDNA or protein contamination. A small transparent yellowish pellet is the best sign of a good RNA prep. I also do two washes with 70% EtOH.
BTW: i wouldn't resuspend the cell pellet in PBS but lyse it directly with the trizol otherwise i would say that you end up with a larger aqueous phase that will influence isopropanol precipitation efficiency.
thanks again tea-test.....
I think you are right....I shouldn't be adding PBS, ...instead I need to add Trizol directly to the cells.
I saw the transparent pellet. I guessed it must have been the RNA pellet.....but it's not yellowish...it's just colorless.....
anyway...thanks a lot. I think I'm on the right path now!
The cloudiness is due to the salts in the PBS precipitating the proteins and probably some of the phenol out of solution.
bob1 on Mar 8 2009, 04:45 PM said:
we don't use phenol....and probably the cloudy interphase is cell membrane, nuclei and other cell or virus parts....I use this method for both cells and viruses
According to manual, white interphase should contain DNA.
Two things. The three layers after addition of chloroform are the bottom clear organic layer, the middle PROTEIN layer, and the top layer of nucleic acids. Secondly, you DO use phenol since it is a component of the TRIzol reagent.
Dr Teeth on Mar 9 2009, 07:09 AM said:
thank you, didn't know that