HELP: RNA isolation from cell cultures

Robert Slany rslany at leland.stanford.edu
Sat Aug 12 14:41:25 EST 1995


Hi Laurie,
ok. sounds to me like a typical phenol extraction failure. In the 
GIT method according to Chromzynski (I have no idea how it is 
spelt correctly) it is extremely important to use phenol which is 
acidic (equilibrated with water only). If your solutions are 
close to neutral or even basic most of the DNA will stay in the 
interphase, whereas it enters the phenol phase when the phenol is 
acidic. Thats why you should spend some time to equilibrate your 
phenol for DNA extractions!!! For RNA extractions of course you 
want exactly that to happen.
Additionally don't mess around with your cells to much after 
lysis. You will break the chromosomal DNA into 1000 pieces and 
you will never get rid of it anymore. Carefully remove the 
supernatant and extract it a couple of times with acidic phenol 
and you should be fine. 
Moreover you should be able to see the contaminating DNA on a gel 
as a high molecular weigth band.

Hope that helps

Robert

Robert Slany, Stanford Medical Center, rslany at leland.stanford.edu




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