cloudy phase during RNA extraction with Trizol
Posted 06 March 2009 - 11:42 PM
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?
Posted 07 March 2009 - 05:41 AM
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.
Edited by tea-test, 07 March 2009 - 06:02 AM.
Posted 07 March 2009 - 10:02 AM
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?
Posted 07 March 2009 - 11:15 AM
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.
Edited by tea-test, 07 March 2009 - 01:21 PM.
Posted 07 March 2009 - 09:13 PM
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!
Posted 08 March 2009 - 03:45 PM
Posted 09 March 2009 - 01:17 AM
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
Posted 09 March 2009 - 03:03 AM
I never trust anything that can't be doubted.
Posted 09 March 2009 - 06:09 AM
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Posted 09 March 2009 - 06:31 AM
thank you, didn't know that
Posted 09 March 2009 - 03:39 PM