DNA precipitation with Am. Acetate

skorycd at mail.ncaur.usda.gov skorycd at mail.ncaur.usda.gov
Fri May 14 12:13:19 EST 1999


Check out Focus article " Ethanol Precipitation; Ammonium Acetate as
an alternative to Sodium Acetate" 1987 9:2

http://www2.lifetech.com/focus_page.html

Christopher Skory
National Center for Agricultural Utilization Research
United States Dept. of Agricultural
Peoria, Illinois

In article <3737d01c.91140422 at news>,
  jrt at home.com (John Thompson) wrote:
> I have a slighty different perspective from Frank.  More than 2
> volumes of EtOH is supposed to increase the proportion of salt that
> coprecipitates.  I haven't verified that but have never found a need
> to go with more than 2 or 2.5 volumes.  Regardless, 70% EtOH washes
> are always recommended to remove excess salt.
>
> I've always preferred ammonium acetate as the counter ion; it's more
> EtOH soluble than sodium. Prepare a 5M stock and add 1/10th vol for
> routine DNA recovery or equal vol. (2.5M final before EtOH addition)
> which is supposed leave protein in the supernatant (again not verified
> personally).  Some enzymes (notably polynucleotide kinase... which is
> not often used anymore) are sensitive to ammonium ions, but an extra
> 70% wash should be sufficient even for such enzymes.
>
> PEG precipitation protocols are also advertised as selective for
> recovery of DNA in the presence of protein and often used to clean up
> dirty DNA (DNA that won't cut).  PEG is pretty inert and I've never
> heard of it interfering in enzyme reactions or transformations myself.
> Again 70% EtOH washes eliminate most if not all the residual PEG
> anyway. So PEG may have some merits but I've never had much need for
> the PEG procedures and doubt they will improve your yield.
>
> Suggestion.... Instead of dumping precipitate supernatant (making
> assumptions here), try using a pipet tip to gently withdraw all but
> the last 50-100 ul to avoid disturbing the pellet.  Let the washes
> take the salt away and then just dry the residual 70% EtOH left at the
> end.
>
> Regards,
> John Thompson
> Merck Research Labs
>
> "Frank O. Fackelmayer" <fof1 at chclu.chemie.uni-konstanz.de> wrote:
>
> >Hi Wilson,
> >In general, EtOH precipitation is considered quantitative, i.e. very
> >close to 100% recovery. When you perform, e.g. EtOH precipitation on
> >radioactively labelled DNA, and then count both the pellet and the
> >alcoholic supernatant, you´ll see only very little radioactivity in
the supe.
> >Most people use 2.5 vol of absolute ethanol for precipitation. There
is
> >no risk in using more (3 to 4 vols are still ok), but, of course,
you´ll
> >have to have a certain minimal concentration of EtOH. I have never
> >checked with 2vols, and I guess that could be the reason for your low
> >recovery. Simply use more EtOH next time. You could also try
isopropanol
> >(add 0.6 vols minimum), but you´ll have to wash your pellet
thoroughly
> >with 70% EtOH to get rid if the isoprop. I would recommend against
PEG,
> >as it coprecipitates and might cause problems in downstream
applications
> >(as does isoprop when it is not removed completely).
> >
> >Frank
> >
> >
> >Wilson wrote:
> >>
> >> Dear all,
> >> Recently, I have encountered a problem that the recovery of DNA
after 2 vol
> >> Ethanol (-20oC cold) with 0.1 vol NaOAc  (pH 5.2, 3 M) was low
(below 50%)
> >> and fluctuated.  I have incubated the DNA for 15 minutes in ice and
then
> >> centrifuged at 13000 rpm at room temp for 15 minutes.  The size of
DNA is
> >> 5.2 kb and amount is about 5 ug.
> >> I would like to know in general case, what is the percentage of the
recovery
> >> of DNA after Ethanol precipitation?  I would like to switch to PEG
> >> precipitation to obtain higher recovery, is the PEG precipitation
can
> >> provide higher DNA recovery?
> >>
> >> Thanks a lot.
> >>
> >> Wilson
>
>


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