Wallace, D.M. 1987. Precipitation of Nucleic Acids

[5] PRECIPITATION OF NUCLEIC ACIDS 41

with concentrated buffers at pH 8 or 9 before extracting DNA. All manipulations 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 extracting. 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 mixture of ACE-saturated phenol and chloroform-isoamyl alcohol as noted above for large-scale RNA extractions.

[5] Prec ip i tat ion of Nucle ic Acids

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 precipitated 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 subjected to ethanol precipitation as these materials will also precipitate along with the nucleic acid; preliminary dialysis of such samples is essential 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 (sample) 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).


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 Tris-HCl at pH 8; 1 mM Na2EDTA).

Notes and Alternatives. A common alternative to using sodium acetate 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 precipitation of deoxyribonucleoside triphosphates (dNTPs) TM and therefore is useful for removing unreacted triphosphates from the products of reverse transcriptase, DNA polymerase, or terminal transferase-catalyzed reactions. 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 increased to permit temperature equilibration (at least 30 min for a 30 ml sample). No matter what the volume being processed, overnight precipitation at -20 is effective even at very low DNA concentrations (10 ng/ml DNA in the original sample). The optimal centrifugation method for precipitate 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).


ties are to be recovered from 1-ml volumes or less in the absence of carder, more rigorous ultracentrifugation must be applied. One such technique 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 tRNA cannot be used, for instance when the DNA is to be phosphorylated 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 centrifugation (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-thaw 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. Shapiro, Anal. Biochem. 110, 229 (1981).


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 intended, 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 subsequent 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 concentration, 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).


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 relatively high purity. Indeed, DNA can be precipitated from solutions containing 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 precipitated 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 spermine 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. McClure, Nucleic Acids Res. 9, 5493 (1981).


5. At this stage a 70% ethanol wash (-20 ) can be performed to remove 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 removal 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 spermine 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 precipitate, 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 precipitation. 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-


taining lower phase will be reduced as water partitions into the butanol phase, thus increasing the DNA concentration. Butanol extraction is carried 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 containing high salt concentrations whereas small RNAs and DNA in general remain soluble.

Differential RNA Precipitation with Lithium Chloride. After preliminary 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 Tris- 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

Wallace, D.M. 1987. Precipitation of Nucleic Acids

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