no waterphase in phenolextraction

David L Haviland David.L.Haviland at uth.tmc.edu
Thu Jun 1 14:38:03 EST 2000


Paul E Wiehl wrote:

> The addition of Chloroform will help the phase separation. The loss of the
> DNA might be due to the pH of your solution. If I recall for RNA
> extractions, the low pH favors the DNA staying in the Interphase.
>                                                 Paul W.
>
> On Tue, 30 May 2000, LoPony wrote:
>
> > Make sure the phenol is saturated with buffer-
> >
> > Try adding 1/4 to 1 vol of chloroform to the prep after mixing the phenol
> > and then centrifuge.  I've had this experience myself in some RNA
> > extractions.  A little chloroform won't hurt anything and adds density of
> > course.

Hi:

These are all good suggestions but the one thing experience taught me was to
use an equal volume to equilibrate.  So if I was prepping a 500g  bottle, I'd
use 500 mls of 2M Tris.  If I used much less than 200-250 mls then yes, the
phenol would simply "slurp" it all up and I had no phase separation.  The
addition of chloroform would often help but having the light come on and using
an equal volume to equilibrate was better. Another item of interest is to do as
few equilibrations as possible.  What I would do here is to use 2M of
un-adjusted (pH) of Tris-Base (Trizma).  Usually, two extractions was all that
was needed to bring the pH up above 7.4.   I would add the 8-hydroxyquinaline
before the equilibration so one knows what phase is what then add the 2-ME when
finished.

Hope this helps,
David
===========
David L. Haviland, Ph.D., Asst. Prof. Immunology
University of Texas - Houston, H.S.C.
Institute of Molecular Medicine, R907
2121 W. Holcombe Blvd.,  Houston, TX  77030
713.500.2413-Voice//713.500.2424-FAX
-----------------
If everything seems to be going so well, you have obviously
overlooked something.
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