Ammonium Acetate Protein Precipitation

kim at m44.unm.edu kim at m44.unm.edu
Fri Jun 26 11:08:05 EST 1992


Dear group:

In a BRL Focus (Vol 9, number 2), there was an article about parameters of
ethanol precipitation using Ammonium Acetate.  In it, there was a description
of a procedure to precipitate proteins from solutions containing protein and
DNA using Ammonium Acetate.   In brief, the solution was made 2.5 M Ammonium
Acetate, centrifuged15 minutes at 16000 x g, the supernatant transferred to a
new tube, and the DNA precipitated by addition of 2 volumes Ethanol.

Does anyone have any direct experience with this procedure?  Does it work?  In
the past year, there have been several "methods" papers in Nucleic Acids
Research describing a NaCl salting-out process for removing proteins from
genomic DNA preps in which saturated NaCl is added to the prep after Proteinase
K digestion to remove proteins and polypeptide fragments, and claims were made
of high 260/280 ratios, high DNA yields, while retaining the ability to use the
recovered DNA as a template for a number of different enzymatic reactions.

I'd like to see if I can eliminate Phenol/Chloroform extraction steps in
preparation of templates for in vitro transcription in making antisense RNA
probes.  Currently, I digest the cut plasmid with Proteinase K, then phenol
extract twice, chloroform extract, then ethanol precipitate.  I'd like to see
if I can Proteinase K digest, then salt out the protein with ammonium acetate
or NaCl, followed by ethanol precipitation.  Is this a dumb idea?

Thanks for reading.

Daniel Kim
KIM at FLOVAX.LANL.GOV



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