[ 5] PRECI PI TATI ON OF NUCLEI C ACIDS 41

with concentrated buffers at pH 8 or 9 before extracting DNA. All manip-

ulations are carried out at room temperature.
Typical yield: where DNA is present at 1/zg or greater the recovery is
over 90%. Indeed, the most significant losses occur during the subsequent
ethanol precipitation (see this volume [5]).
Special Cases. $1 nuclease products require that the pH be altered
prior to phenol extraction by adding Tris base to a final concentration of
50 mM. Dephosphorylation reactions require more than one extraction to
remove bacterial alkaline phosphatase completely (three extractions are
recommended). Many investigators treat samples with proteinase K at a
final concentration of 50-200/~g/ml for 10 min at 37 before phenol ex-
tracting. As noted above, extraction of oligodeoxyribonucleotides may
require salt to drive the oligomer into the aqueous phase.
Small-scale RNA extractions are performed similarly with a 1 : 1 mix-
ture of ACE-saturated phenol and chloroform-isoamyl alcohol as noted
above for large-scale RNA extractions.
[5] Pr eci pi t at i on of Nucl ei c Aci ds
By DONALD M. WALLACE
During the course of a cloning project many occasions occur when it is
necessary to concentrate nucleic acid samples or change the solvent in
which a nucleic acid is dissolved. Fulfillment of these requirements is met
by nucleic acid precipitation techniques.
Ethanol Precipitation
Ethanol precipitation is probably the most versatile method of concen-
trating nucleic acids and is commonly used to recover DNA or RNA after
extraction from biological sources or to retrieve phenol-extracted DNA
products of in vitro enzymatic manipulations (see this volume [4]).
In brief terms, a precipitate is formed by leaving a mixture of the
sample, salt, and ethanol at low temperature ( - 20 or lower). The precipi-
tated salt of the nucleic acid is then sedimented by centrifugation, the
ethanol supernatant removed, and the nucleic acid pellet resuspended in a
buffer appropriate to its subsequent use. The choice of salt used for the
precipitation is determined both by the nature of the sample and by the
intended use of the nucleic acid.
Copyright 1987 by Academic Press, Inc.
METHODS IN ENZYMOLOGY, VOL. 152 All rights of reproduction in any form reserved.
42 ISOLATING AND CHARACTERIZING NUCLEIC ACIDS [5]
Samples containing phosphate or 10 mM EDTA should not be sub-
j ect ed to ethanol precipitation as these materials will also precipitate
along with the nucleic acid; preliminary dialysis of such samples is essen-
tial before ethanol precipitation.
[Editors' Note. When nucleic acids are precipitated solely to change
solvents, one might consider drop dialysis. 1 Drop dialysis represents a
practical alternative to conventional dialysis in bags for volumes of 50/zl
or less. Float a Millipore (Bedford, MA, catalog no. 01300, VSWP)
0.025/.tm porosity filter disk, 13 mm diameter, shiny side up, on the sur-
face of 5 ml of the diffusate (the solution one is dialyzing against). A 35-
mm petri dish is a convenient vessel. Carefully pipet the retentate (sam-
ple) onto the center of the floating disk; it should form a dome-shaped
drop. After 2-3 hr, a sample containing approximately I /~g mRNA
should be recovered from the disk with >90% yield and should be found
virtually free of labeled triphosphates, salt, or traces of phenol. Since a
steady hand is required, a few practice tries with a solution similar to the
sample are recommended.]
Basic Method of DNA Precipitation with Ethanol
For recovery of DNA from a typical reaction (e.g., for l / zg DNA in
20 izl):
1. To 20/A aqueous DNA sample in a plastic microcentrifuge tube add
2/~l 3 M sodium acetate, pH 5.5 (i.e., 0.1 volume giving 0.3 M Na+), and
40/ A ethanol (i.e., 2 volumes).
2. Mix well by vortexing and immerse the tube into a - 70 bath com-
posed of methanol plus dry ice for 15 min. Powdered dry ice can be
substituted for the bath. The mixture will freeze or form a slurry in this
time.
3. Centrifuge the DNA precipitate in a bench-top microcentrifuge at
maximum speed for 10 min (cold room). A whitish pellet of DNA should
appear at the bottom of the tube. In general, pellets of 10/zg are visible
whereas pellets of 2/~g will be invisible.
4. Remove the ethanol supernatant using a micropipettor (Rainin Pi-
petman or the equivalent), taking care not to disturb the pellet or the area
of the tube where the pellet should be located.
5. Add 100/zl 70% ethanol ( - 20 ) to the sample and vortex. This step
removes any solute trapped in the precipitate.
6. Resediment the precipitate by centrifugation for 2 min, and remove
the supernatant as before.
R. Marusyk, Anal. Biochem. 105, 403 (1980).
[5] PRECIPITATION OF NUCLEIC ACIDS 43
7. Dry the pelleted DNA for 1-2 min in a lyophilizer (Speed Vac
Concentrator, Savant Instruments Inc.) or vacuum desiccator taking care
to release the vacuum gently so as not to dislodge the dried sample.
8. Resuspend the DNA in TE (pH 8) buffer (10 mM Tri s-HCl at pH 8;
1 mM Na2EDTA).
Notes and Alternatives. A common alternative to using sodium ace-
tate at 0.3 M is to make the sample 2.5 M in ammonium acetate prior to
the addition of ethanol (add 0.5 volume 7.5 M ammonium acetate followed
by 2.5 to 3 volumes of ethanol). Cool for 10 min in dry ice or at -70; the
sample will freeze slowly or not at all if left for hours. Besides adequately
substituting for the sodium salt, this method also prevents the precipita-
tion of deoxyribonucleoside triphosphates (dNTPs) TMand therefore is use-
ful for removing unreacted triphosphates from the products of reverse
transcriptase, DNA polymerase, or terminal transferase-catalyzed reac-
tions. Typically, two serial precipitations result in the removal of about
99% of the dNTPs, and greater than 90% DNA recovery. However, if the
DNA is to be phosphorylated or tailed, ammonium acetate should be
avoided because ammonium ions inhibit the enzymes required for these
processes.
Another alternative makes use of Li as the cation. To the solution
containing DNA or RNA, add 0.1 volume 8 M LiCI (filter sterilized) and 2
to 3 volumes of ethanol. Since LiCI is highly soluble in ethanol-containing
solutions at - 70 , salt is not coprecipitated with the nucleic acid.
Should the original DNA sample also contain sodium dodecyl sulfate
(SDS), the detergent is most effectively removed by making the sample
0.2 M with sodium chloride before ethanol addition (usually by adding
0.04 volume 5 M NaC1 followed by 2 volumes ethanol). SDS remains
soluble under these conditions.
For small volumes of 1 ml or less containing at least 10/xg/ml DNA in
the original sample, the 15-min precipitation at - 70 gives at least 80%
recovery. If larger volumes are to be handled, the precipitations can be
carried out in 15-30 ml glass Corex tubes and the period at - 70 in-
creased to permit temperature equilibration (at least 30 min for a 30 ml
sample). No matter what the volume being processed, overnight precipi-
tation at - 20 is effective even at very low DNA concentrations (10 ng/ml
DNA in the original sample). The optimal centrifugation method for pre-
cipitate recovery is dictated by the volume of the sample and the quantity
of the DNA present. For precipitates of at least 1/xg in 1 ml or less the
bench-top microcentrifuge method is adequate. When nanogram quanti-
la H. Okayama and P. Berg, Mol. Cell. Biol. 2, 161 (1982).
44 ISOLATING AND CHARACTERIZING NUCLEIC ACIDS [5]
ties are to be recovered from 1-ml volumes or less in the absence of
carder, more rigorous ultracentrifugation must be applied. One such tech-
nique is that of Shapiro, 2 by which nucleic acid present at I0-1000 ng/ml
is recoverable at 70-100% efficiency. The precipitated DNA samples in
completely filled microcentrifuge tubes (to prevent cracking) are placed in
the buckets of a swing-out rotor (e.g., 0.4-ml tubes in an SW41 bucket;
1.4-ml tubes in an SW27 bucket) which are half-filled with ice-cold 20%
ethanol (to prevent freezing). The samples, floating inside the ultracentri-
fuge buckets, are subjected to centrifugation of 41,000 rpm for 30 min
(SW41), or 27,000 rpm for 60 min (SW27) at - 2 . The pelleted DNA
present at the bottom of the microcentrifuge tube is treated as already
stated, except that the centrifugation after the 70% ethanol wash should
be carded out in a swing-out rotor in a refrigerated machine (e.g., HB-4
rotor in a Sorvall RC-5B centrifuge at 11,500 rpm for 15 min at -10). It
should be noted that in most situations nanogram DNA quantities can be
recovered using the basic protocol as long as a coprecipitant is also used,
for example 50/zg/ml tRNA. However, in circumstances when carrier
t RNA cannot be used, for instance when the DNA is to be phosphory-
lated using polynucleotide kinase, ultracentrifugation may be the only
method of recovering DNA as a precipitate in ethanol. When dealing with
picogram DNA amounts, the use of coprecipitants (tRNA or purified
glycogen) is essential for effective DNA recovery.
If larger volumes of material are being handled, high-speed centrifuga-
tion (10,000 rpm for 30 min in an HB-4, SS-34, or GSA rotor in a Sorvall
RC-5B centrifuge at - 10 to 0 ) is adequate for 10 /xg/ml quantities of
nucleic acid, whereas ultracentrifugation (40,000 rpm in an SW41 rotor at
0 ) should be used for lower concentrations.
The methods mentioned are suitable for DNA larger than 200 base
pairs; for improved recovery of smaller DNAs the sample should be made
10 mM in magnesium chloride before ethanol precipitation.
DNA is best stored at 4 in TE (pH 8) buffer. The EDTA chelates
heavy metal ions which are commonly required for DNase activity, while
the use of pH 8 minimizes deamidation. For very long term storage (5
years or more) DNA can either be frozen at - 70 and not subjected to any
freeze-t haw cycles, or it can be dissolved in buoyant CsC1 at 4 .
RNA Precipitation with Ethanol
The method for precipitation of RNA is virtually identical to that for
DNA. For a 10-ml aqueous RNA-containing sample add l ml (0. I volume)
2 O. J. Shapi ro, Anal. Biochem. 110, 229 (1981).
[5] PRECIPITATION OF NUCLEIC ACIDS 45
3 M sodium, potassium, or ammonium acetate (pH 5.5) or 0.1 volume 8 M
LiC1 followed by 25 ml (2.5 volumes) ethanol. Precipitate and treat as
described for DNA precipitation by ethanol. Resuspend the dried RNA
pellet in a suitable volume of sterile water or appropriate buffer, and store
at - 70 , or in liquid nitrogen vapor.
Notes. For milliliter volumes, precipitation is normally carried out
overnight at - 20 followed by centrifugation in 30-ml glass Corex tubes at
10,000 rpm for 30 min (HB-4 or SS-34 rotor in a Sorvall RC-SB centrifuge
at - 10 to 0).
The choice of salt used is dictated by the intended use of the RNA. For
instance, if oligo(dT)-cellulose chromatography in the presence of SDS is
to be performed, the sodium salt is chosen. If cell-free translation of
mRNA is to be carried out, potassium acetate is used. Ammonium acetate
can substitute for the potassium salt in this situation. Note that chloride
should not be used when translation in cell-free systems is intended, since
C1- interferes with initiation of protein synthesis. When the RNA to be
precipitated also contains SDS and efficient cell-free translation is in-
tended, at least two serial precipitations are required: the first with Na to
precipitate the RNA, leaving the SDS in solution, and the second with K
to replace the sodium ion in readiness for translation. Li should be
avoided when the precipitated RNA is to be reverse transcribed.
The duration and temperature of precipitation, the centrifugation
methods of precipitate recovery, and the concentration limits are essen-
tially the same as those stated for DNA ethanol precipitations. However,
because the original RNA level is not always known, for example when
isolating RNA from a biological source, overnight precipitation at - 20 is
recommended.
Storage of RNA in aqueous solution at -70 is only recommended if
the sample is not going to be subjected to repeated freezing and thawing.
Storage of the sample in aliquots at suitable volumes can partially circum-
vent this problem. Also, the addition of a drop of 0.2 M vanadyl-ribonu-
cleoside complexes (VRC) to the RNA prior to freezing will prevent
RNase-catalyzed RNA degradation. VRC does not interfere with the sub-
sequent use of the RNA unless cell-free translation is intended, in which
case the VRC can be removed by phenol extraction (translation of mRNA
by microinjection into Xenopus oocytes is unaffected by a moderate con-
centration, e.g., 5 mM of VRC). 3 Poly(A) mRNA can be stored for years
by this method. Alternatively, RNA can be stored securely as an ethanol
precipitate at - 20 .
3 R. S. Puskas, N. R. Manley, D. M. Wallace, and S. L. Berger, Biochemistry 21, 4602
(1982).
46 ISOLATING AND CHARACTERIZING NUCLEIC ACIDS [5]
Note that all buffers, glassware, and plasticware must be rendered free
of RNase before working with RNA (see this volume [2]).
Isopropanol Precipitation of DNA
Isopropanol-induced precipitation of DNA minimizes the total volume
of the precipitating sample. The precipitation is carried out by adding an
equal volume of isopropanol (2-propanol) to the sample, and treating the
mixture in the same way as for ethanol precipitations. The drawbacks of
this method are that 2-propanol cannot be easily lyophilized due to its
relatively low volatility, and also that salts present in the original sample
tend to coprecipitate with the DNA. Because of these problems it is
common practice to carry out an ethanol precipitation of the sample im-
mediately following a 2-propanol precipitation.
Spermine Precipitation of DNA
The precipitation of DNA by the polyvalent cation spermine 4 is useful
for the recovery of DNA from dilute solutions (0. I - 100/~g/ml). Due to the
selective nature of the precipitation, the procedure yields DNA of rela-
tively high purity. Indeed, DNA can be precipitated from solutions con-
taining proteins; for example, restriction enzyme digests containing bo-
vine serum albumin (BSA) retain 4-5% of the initial BSA in the DNA
pellet, and bacterial cleared lysates retain 6-8% of the protein as a con-
taminant in the recovered DNA. Nucleoside triphosphates are not precip-
itated by spermine, thus providing a method of recovering dNTP-free
DNA; two sequential precipitations are recommended.
For spermine precipitation of a 50-~1 sample containing moderate salt
typical of genetic engineering reactions (e.g., 0.1 M KCI, 10 mM MgCI2, 1
mM dithiothreitol, 10 mM Tris-HCl at pH 8):
1. To the sample in a 1.4-ml microcentrifuge tube, add 5 /zl 0.1 M
spermine tetrahydrochloride (0.1 volume, resulting in 10 mM final sper-
mine concentration).
2. Mix well by vortexing and place in ice for 15 min to form the
precipitate.
3. Centrifuge the precipitate in a bench-top microcentrifuge for 10
min. For nanogram quantities of DNA, centrifugation at 10,000 rpm for 15
min in a high-speed instrument is required.
4. Remove the supernatant using a micropipettor, taking care not to
disturb the pellet.
4 B. C. Hoopes and W. R. McCl ure, Nucleic Acids Res. 9, 5493 (1981).
[5] PRECIPITATION OF NUCLEIC ACIDS 47
5. At this stage a 70% ethanol wash ( - 20 ) can be performed to re-
move any trapped solute (see ethanol precipitation for method).
For removal of the spermine add 0.2 ml cation-exchange buffer in ethanol
(1 part 0.3 M sodium acetate or potassium acetate, 10 mM magnesium
acetate to 3 parts ethanol; v/v) and vortex to disperse the pellet thor-
oughly. After leaving in ice for 1 hr with periodical mixing, the sample is
subjected to centrifugation as before and the supernatant removed. The
pelleted DNA is then treated in the same manner as that stated for ethanol
precipitations.
Notes. Because the efficiency of spermine precipitation is sensitive to
the ionic strength of the sample, the amount of spermine to be added is
determined by the salt content of the sample. At 0.1 mM spermine, DNA
will precipitate in the absence of salt. However, if the DNA solution also
contains 10 mM EDTA, 6 mM MgCI2, and 10 mM KCI, for example, the
final spermine concentration must be increased 10-fold. Precipitation of
DNA dissolved in 0.1 M KC1 or in 0.1 M KCI, 10 mM MgCI2 requires a
final spermine concentration of 2 or 10 mM, respectively.
An alternative method of spermine removal for large amounts of DNA
is to dissolve the pellet in a small volume of buffer containing 0.5 M salt
and to dialyze against the same buffer (two changes) for 24 hr. The re-
moval of salt from the concentrated DNA sample can then be achieved by
conventional ethanol precipitation, or by dialysis against the desired
buffer (e.g., TE, pH 8).
Besides precipitating DNA from dilute solutions and from complex
mixtures, another application is the differential precipitation of relatively
high-molecular-weight DNAs. Under moderate salt conditions the sper-
mine method preferentially precipitates DNA of 0.2 kilobase (kb) and
larger; material shorter than 60 base pairs (bp) does not precipitate. A
direct application of this phenomenon pointed out by the originators 4 is
the purification of linker-bearing high-molecular-weight DNA as a precipi-
tate, by selective removal of the oligodeoxyribonucleotide linker debris
generated after restriction endonuclease digestion.
Butanol Concentration of DNA
DNA can be recovered from dilute solutions by extracting several
times with butanol to concentrate the sample prior to ethanol precipita-
tion. An equal volume of 2-butanol is mixed vigorously with the sample
followed by phase separation on a bench-top microcentrifuge for 1 min
(for microliter volumes). The upper organic layer is carefully removed
with the aid of a micropipettor. The volume of the aqueous, DNA-con-
48 ISOLATING AND CHARACTERIZING NUCLEIC ACIDS [ 5]
taining lower phase will be reduced as water partitions into the butanol
phase, thus increasing the DNA concentration. Butanol extraction is car-
ried out until the desired final volume of sample is attained. Because this
procedure also concentrates the salts present in the sample, a final ethanol
precipitation is carried out and the pelleted DNA resuspended in the
desired buffer.
Precipitation of RNA with High Salt Concentrations
Large RNAs (rRNA, hnRNA, mRNA) are insoluble in solutions con-
taining high salt concentrations whereas small RNAs and DNA in general
remain soluble.
Differential RNA Precipitation with Lithium Chloride. After prelimi-
nary extraction of RNA from a biological source by phenol extraction or a
guanidine-based technique the large RNA can be separated from contami-
nant low-molecular-weight DNA and RNA (tRNA and 5 S rRNA) by
subjecting the aqueous sample to LiC1.
1. To the sample add 1 volume of 8 M LiCI.
2. Mix vigorously and incubate at - 20 for at least 2 hr.
3. Sediment the precipitated RNA at 10,000 rpm for 10 min at 0 (e.g.,
GSA or SS-34 rotor in a Sorvall RC-5B at 0). Remove the supernatant
fluid; it contains small RNAs.
4. Resuspend the pellet in water at about 1 mg/ml and repeat the
procedure. Finally, resuspend the purified RNA in water and subject it to
two conventional ethanol precipitations. (To recover the small RNAs,
add 2-3 volumes of ethanol to the LiCI supernatant fluid and process
according to conventional ethanol precipitation procedures.)
Should high-molecular-weight DNA contaminate an RNA sample, treat
with RNase-free DNase (25 lzg/ml DNase in 10 mM MgC12, 50 mM Tri s-
HC1 at pH 7, at 37 for 30 min) followed by phenol extraction and ethanol
precipitation. A simple way for doing this is to add VRC, the RNase
inhibitor, to the DNase. The VRC at 10 mM protects the RNA from
RNase without affecting the activity of the DNase. 3