Hot/Acid Phenol Extraction

Bruce R. Troen bruce.troen at med.umich.edu
Sat Dec 25 13:23:12 EST 1993


Mike -

     We make lots of RNA from mammalian cells in culture and use acid
phenol extraction with guanidinium isothiocyanate and then chloroform. 
We prepare the phenol by equilibrating it with deionized water and
don't even both adjusting pH.  I believe the extraction works because
the acidic pH permits a selective extraction of the RNA into the
aqueous phase (though this is not absolute).  There are many protocols
that use this procedure (lots recently published in BioTechniques). 
There are also several different commercial preparations of the GTC and
phenol available.  We make our own.

     If you need to be absolutely sure there is no DNA in the sample,
then you should treat with DNAase (for example, differential display -
PCR requires this).  However we perform RT-PCR without any DNAase
treatment.  Of course we also design our primers so that we can discern
if the amplified fragment contains extra material such as an intron.

Hope this helps.

Bruce R. Troen, M.D.
University of Michigan Medical School



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