ethanol precipitation
Basavareju Shankarappa
>>precipitation of salt specially after prolonged incubation at
>>-20 C. I learnt this from my experience rather than anything.
>0.2 M NaCl is high enough, but at least 0.5 M will NOT precipitate
>when two volumes of EtOH are added, even after extended chilling
>at -20 deg. If you see salt precipitating, there must be something
>else in your preparation. (Note that ammonium acetate is both
>more soluble and less effective than NaCl at leading to DNA
>precipitation, undoubtedly due to the larger ion sizes, so
>considerably higher concentrations of AmOAc are used.)
>>I have also learnt that this salt pptn is very very hard to get
rid off.
>If salt does precipitate, it can easily be removed by washing with
>70% EtOH. The DNA will not dissolve at room temperature, but the
>salt will. (This can also be used to remove CsCl. I find a few
>washes with 70% EtOH is easier than dialysis when I have to
>remove CsCl from a plasmid prep. NOTE: do NOT chill when
>removing CsCl, which has a high temperature coefficient of
>solution.)
My problems with salt precipitation were in the isolation of
genomic DNA primarily from bacterial species and not with CsCl
preps. This happened awhile back and as I remember I started out
with a bacterial suspension that contained about 150 mM NaCl for
the purpose of maintaining isotonicity. I think I added sodium
acetate before adding ethanol. May be because of the presence of
two different salts the salt pptd out. The funny thing was when I
dissolved the whole precipitate in Distilled water and tried to
reprecipitate it; Even though I did not add any additional salt
(Sod acetate in this case) the salt precipitate formed again. I
finally gave up and started from a new batch where I did not add
any salt and managed to obtain good quality DNA.
Did anybody else face similar circumstance? I think this may also
have something to do with the lipopolysacharide content of
bacterial species. So I think Bill is right in that it might have
been something in my sample. Although this was long back, I am not
doing the same work, and have not had the same problem anymore, I
am still curious why it did happen.
The problem I have with the use of ammonium acetate is the
requirement for the addition of two volumes of salt in addition to
the ethanol. This usually screws up the maximum volume that I can
handle in a 1.5 ml tube. Any suggestions for avoiding this, like
using higher concentration of amm. acetate?
Thanks for the many postings.
Raj Shankarappa
Peter B. Berget
addition of only 1/20th volume to get to the 0.4 M concentration we use
for 2 volume ethanol precipitations.
Peter B. Berget
Department of Biological Sciences
Carnegie Mellon University
4400 Fifth Avenue
Pittsburgh, PA 15213-3890
Bill Melchior, NCTR/FDA
>addition of only 1/20th volume to get to the 0.4 M concentration we use
>for 2 volume ethanol precipitations.
It's nice to know that 0.4 M is sufficient to precipitate DNA; since the
ammonium ion is larger (ie lower charge/volume) than Na+, I would have
thought it would take a higher concentration.
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.
________________________________________________________________________________
The opinions stated are mine, not those of NCTR or its sponsoring organizations.
Bill Melchior || "You have lawyers the way
National Center for Toxicological Research || other people have mice."
Jefferson, AR 72079 ||
(501) 543-7206 || -Kenneth Duncan, English
|| Health & Safety Executive,
WMEL...@NTDOC.NCTR.FDA.GOV || to US regulators
Mike Poidinger
Am ions severely inhibit polynucleotide kinase,
are there any other salts which have similar effects
on other commonly used enzymes? I include in this
category salts which co ppt with the DNA and thence inhibit enzymes.
BTW, I only ever use NaAc for ppting DNA
Mike Poidinger
Dept of Microbiology
University of Reading
"Spam, spam, spam, kits and spam"
bhj...@triton.unm.edu
>
>The problem I have with the use of ammonium acetate is the
>requirement for the addition of two volumes of salt in addition to
>the ethanol. This usually screws up the maximum volume that I can
>handle in a 1.5 ml tube. Any suggestions for avoiding this, like
>using higher concentration of amm. acetate?
>
enough to precipitate 10 minipreps. You just add 2ul of tube 2
to 100 ul of tube 3...
:-)
Brian
Robert Coelen
removal of single nucleotides (and I think also small oligos).
Certainly 2 successive etoh pptns with amm OAc as salt will remove in
excess of 95% of single nucleotides.
Robert Coelen
Dept of Microbiology
UWA
"Yuk, who would eat spam !"
the End
>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.
Cheers,
Jim Graham
Biology/Chemisrty Depts.
Indiana University
Andrew F. Cockburn
>>>I have observed that using NaCl for pptn of DNA often results in
>>>precipitation of salt specially after prolonged incubation at
>>>-20 C. I learnt this from my experience rather than anything.
Flame on.
My answer is simple: you shouldn't be precipitating DNA at -20 C (or
worse yet, at -80 C). DNA is insoluble in 60% ethanol at 4 C (with
the proper salt concentration). It doesn't become "more insoluble"
at lower temperatures, this is impossible. The idea that nucleic
acids precipitate faster at low temperatures is a myth.
The process of insoluble DNA aggregating to form precipitates is a
chemical reaction, and like other chemical reactions occurs more
rapidly at higher temperatures. Putting it in the freezer SLOWS the
precipitation, it does not speed it up.
BTW, BRL tested this seven or eight years ago and published the results
is Focus. Overnight precipitation at 4 C was the best. The most
critical factor in recovery was how long and hard the centrifuge
spin was.
Flame off.
Andrew Cockburn
USDA/ARS/MAVERL
Disclaimer: Of course, my grad students put their DNA into the freezer
to precipitate...
wets...@wums.wustl.edu
guide to molecular cloning", 2nd Ed. in which he states that temperature has
no effect on the recovery of DNA in an EtOH precipitation. What DOES make a
major difference is the time of centrifugation. 30 mins at room temp is a
whole lot better that 10 minutes at 4'C when it comes to percentage of DNA
recovered. I beleive the figure was something like 90-95% recovery with a
30 minute spin.
David
ryb...@uctvax.uct.ac.za
> In article <920715220...@phobos.med.pitt.edu>, b...@MED.PITT.EDU (Basavareju Shankarappa) writes:
>>>>I have observed that using NaCl for pptn of DNA often results in
>>>>precipitation of salt specially after prolonged incubation at
>>>>-20 C. I learnt this from my experience rather than anything.
> Disclaimer: Of course, my grad students put their DNA into the freezer
> to precipitate...
Why do they do that? So ANNOYING!! I used to think it was because older
protocols had it in, but I now think it's because it's the lab equivalent of an
urban legend, and the buggers don't ever bother to go and look up methods
properly.
_________________________________________________________________________
| | |
| Ed Rybicki | Now you've got the hang of it |
| Dept Microbiology | There's nothing you can't do with it |
| University of Cape Town | If you're very into it |
| PB Rondebosch 7700 | You can't go wrong.... |
| South Africa | |
| fax 27 21 650 4023 | - Mad John |
| e...@micro.uct.ac.za | (Ogden's Nut-Gone Flake, Small Faces) |
|_________________________________|_______________________________________|
Gabriel Trueba
probes for Southern blot analysis. I wonder whether somebody
in the net has experimented lack of hybidization of PCR products
We would appreciate any suggestion.
Thank you
Gabriel Trueba
Robert Horton
--
Bob Horton /\ "Crash programs fail because of the theory that
U. of Minnesota, CBS || with nine women pregnant you get a baby a month"
1479 Gortner Ave. /||\ -Werner von Braun. Disclaimer:"Bob who?"
St. Paul, MN 55108 ^^ hor...@molbio.cbs.umn.edu/(612) 624-3790