ethanol precipitation (NH4CH2COOH cleaner minipreps)
jgraham at bronze.ucs.indiana.edu
Sun Jul 19 02:57:09 EST 1992
In <920716110344.202003a1 at ntet.nctr.fda.gov> WMELCHIOR at NTET.NCTR.FDA.GOV (Bill Melchior, NCTR/FDA) writes:
>I wonder, though, whether one gets all the benefit of an AmOAc
>precipitation at this low concentration. If I add AmOAC to a crude
>plasmid prep at a final conc of 2.5 M, the solution frequently gets
>cloudy with impurities that I then remove by centrifugation prior
>to precipitation of the DNA. Alternatively, if one redissolves a
>crude prep in 2.5 M AmOAc, the DNA dissolves but many impurities don't,
>again providing for another stage of purification. I suspect that 0.4 M
>might not be so discriminating.
Bill's point here is worth repeating. As well as being a better
counter-ion for nucleic acid precipitation with alcohol, ammonium
acetate alone efficiently precipitates proteins and large RNAs from a
bacterial cell lysate. When used as he describes, very clean minipreps
can be obtained, suitable for double strand sequencing and reportedly
other sensitive procedures.
To incorporate this technique into an alkaline-lysis miniprep,
substitute 7.5 M amonium acetate for 3 M sodium acetate in the step
where you neutralize the alkaline lysate. It is not necessary to
adjust the pH of the 7.5 M stock in my hands. If you are working with
a regular 1.5 ml miniprep, resuspend the cells in 200 ul of
lysozyme-buffer, lyse with 400 ul of NaOH-SDS, and neutralize with 300 ul
of amonium acetate. Percipitate with 0.6 vol Isopropanol (400 ).
If you would like to further reduce the protein and RNA
content of your sample, resuspend the pellet in 2.5 M amonium acetate
and recover the material in the supernatant with a second alcohol
precipitation. Contaminants which are carried through a
regular sodium acetate miniprep and a phenol-chloroform extraction can
be easily seen in a 2.5 M percipitation. If yield is more imporant
than qualtiy, skip the second stage, as it will reduce your yield in
the neighborhood of 40%, depending on the sample.
A final note, do not be disturbed by visible material when
resuspending the initial isopropanol pellet. An insoluble precipitate
can be seen in most preps.
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