Mechanism for acidic phenol prep. of RNA?
dbaggott at reed.edu
Mon Oct 23 12:42:20 EST 1995
Speaking of mechanisms, can anybody explain to me why DNA (chromosomal as
well as plasmid) partitions to the organic phase during extraction with
water-saturated phenol (pH ~5) while RNA (ds as well as ss) stays in the
aqueous? I suspect, but have never tested, that incubating on ice prior
to separating the phases is crucial for this to work. I'm thinking in
particular of the protocol from Current Protocols for preparation of yeast
RNA (which involves suspending cells in a SDS/tris/EDTA solution, adding
acidic-phenol, incubating at 65 for >30min with occasional vortexing, on
ice for 5 min, spin/recover aqueous, re-extract with acidic-phenol with 5
min ice incubation prior to separating phases, clean up aqueous with a
final chloroform extraction and precipitate).
Any insights on this mysterious (to me) process would be appreciated.
In regards to using acid-phenol to remove nicked or linearized DNA from
circular dsDNA, jencsa at net.sote.hu (Jeney Csaba) wrote:
> We did it several times, and it have proved unreliable. One time is perfect, otherwise even the
>superhelical fraction can be lost. I have tried to modify, track down but nothing concludible
>have been found. The major problem that the references do not agree upon the basic protocol,
>for intance one stricty intruct to remove the last traces of proteins and RNAs, the other one
>notice nothing about this staff. And the mechanism is even more obscure there is no two
>Otherwise if anybody has a good experience on this steam building field I love to try it.
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