HELP: RNA isolation from cell cultures
rslany at leland.stanford.edu
Sat Aug 12 14:41:25 EST 1995
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 Slany, Stanford Medical Center, rslany at leland.stanford.edu
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