ANALYTICAL BIOCHEMISTRY 185,230-234 (1990)

A Bacteriophage X DNA Purification Procedure Suitable

for the Analysis of DNA from either Large or
Multiple Small Lysates
Trevor J. Lockett
CSIRO Division of Biotechnology, Laboratory for Molecular Biology, P.O. Box 184,
North Ryde, New South Wales,2113, Australia

Received August 15,1989

for reproducibly efficient digestion by restriction endo-

A method for the efhcient preparation of high quality nucleases, particularly EcoRl. Of these, the DE-52 chro-
bacteriophage X DNA from cleared lysates is described. matography method has the disadvantage that columns
Advantages of the method include high DNA yields tend to become clogged when large volumes of lysate are
(typically around 0.8 ag of DNA/l ml of cleared lysate), processed, while the alkaline treatment step of the am-
speed of processing (approximately 2 h from lysate to monium sulfate procedure, which is essential to ensure
DNA), economy, and the absence of any requirement that the resultant DNA can be cut by restriction endo-
for phenol or chloroform extractions. The technique in- nucleases, leads to some reduction in overall yield and
volves the concentration of phage particles by standard the DNA still requires extraction with phenol and chlo-
polyethylene glycol precipitation followed by enzy- roform (7).
matic treatment to remove contaminating RNA and This paper describes a method for the rapid purifica-
DNA. Phage particles are then lysed with sodium dode- tion of high quality X DNA from either plate or liquid
cyl sulfate (SDS) at elevated pH and temperature. Con- lysates. Although originally developed for the prepara-
taminating protein/SDS complexes are rendered insol- tion of DNA from minilysates it has proven equally use-
uble by the addition of potassium acetate and removed ful for larger scale applications. The key features of the
by centrifugation. The quality of the resultant DNA is method are the purification of bacteriophage particles,
comparable to that prepared by cesium chloride band- removal of contaminating nucleic acids by enzymatic di-
ing for all standard molecular biological purposes pro- gestion, the lysis of phage particles and inactivation of
viding that spermidine is included in all restriction en- nucleases by treatment with SDS’ at elevated pH and
donucleases digestions. o loso Academic PI-, IW. temperature, and the use of potassium acetate to precipi-
tate the resultant protein/SDS complexes. While a
method employing SDS and potassium acetate in the
preparation of DNA from plate lysates of bacteriophage
Derivatives of the bacteriophage X provide a highly X has been described previously, DNA prepared in this
efficient and versatile set of cloning vectors for many way was heavily contaminated with host nucleic acids
molecular biological purposes (l-3). The standard (8) limiting its use to restriction analysis and Southern
method of DNA preparation involving CsCl gradient pu- blotting. However DNA prepared by the modified proce-
rification of virus followed by repeated phenol extrac- dure described here is sufficiently pure for all common
tions (4,5) and ethanol precipitation of the nucleic acid molecular biological purposes without further require-
is expensive, is slow, is cumbersome, produces DNA in ment for extraction with organic solvents.
low yields, and is therefore inconvenient for the analysis MATERIALS AND METHODS
of the large numbers of positive clones which result from Enzymes and Reagents
primary screens of genomic or cDNA libraries. DNase 1, RNase A, spermidine, diethyl pyrocarbo-
Of the numerous other methods for X DNA purifica- nate, and agarose were all purchased from Sigma (St.
tion, only those involving chromatography on cellulose
DE-52 (6) and ammonium sulfate precipitation of the 1 Abbreviations used: SDS, sodium dodecyl sulfate; PEG, polyethyl-
bacteriophage (7) result in preparations sufficiently pure ene glycol6000.

230 0003~2697/90 $3.00

Copyright 0 1990 by Academic Press, Inc.
All rights of reproduction in any form reserved.
 DNA PURIFICATION FROM
Louis, MO). Restriction endonucleases and Xc1857DNA
were obtained from Pharmacia (Uppsala, Sweden). Bac-
terial culture medium was purchased from Difco (De-
troit, MI). Polyethylene glycol 6000 (PEG) was pur-
chased from BDH/Merck (Poole, England). All other
chemicals were AR grade and purchased from Ajax
Chemicals (Australia).
Preparation of Bacteriophage DNA from Minilysates
1. Plate lysates. Recombinant plaques were picked
into 0.3 ml of PSB and phage eluted at 4°C for between 2
and 16 h. An overnight culture of Escherichia coli strain
LE392 was grown in LB, 0.1% maltose and 0.1 ml of this
was added to 0.1 ml of the phage eluate. Phage were per-
mitted to adsorb to the cells for 10 min at 37°C then the
mixture was plated in soft agarose (or agar) on go-mm-
diameter LB agarose (or agar) plates containing 10 mM
MgSO,. Plates were incubated until the plaques merged
(7-16 h) and then flooded with 4.0 ml of 10 mM Tris-
HCl, 10 mM MgS04, 100 mM NaCl, pH 7.5 (PSB). Phage
were permitted to leach into the buffer for 2-16 h at 4°C
with gentle agitation. Buffer was removed from the
plates and cleared by centrifugation at 12,000g for 10
min at 4°C. To 3.0 ml of the supernatant was added 1.8
ml of 20% PEG, 2.5 M NaCl and phage were precipitated
on ice for 30 min.
Precipitated bacteriophage were collected by centrifu-
gation at 12,000g for 10 min at 4°C. The supernatant was
discarded and the tube recentrifuged for 1 min to permit
the last of the PEG containing supernatant to be re-
moved. Precipitates were resuspended in 0.25 ml of PSB
and transferred to l&ml microcentrifuge tubes; RNase
A and DNase 1 added to final concentrations of 20 and
5 Kg/ml, respectively; and the tubes incubated at 37°C
for 30 min. After nuclease digestion, an equal volume of
0.3 M Tris-HCl, 100 mM EDTA, 1.25% SDS, pH 9.0, was
added and tubes were incubated a further 10 min at
65°C. To each tube was added 0.25 ml of ice-cold 3 M
potassium acetate, pH 4.8, and the resultant solutions
were incubated on ice for 5 min. Insoluble material was
removed by centrifugation at 15,000g for 2 min at room
temperature and most of the supernatant (0.7 ml) re-
moved taking care not to include any remaining insolu-
ble flocculent material. Isopropanol (0.5 ml) was mixed
with each supernatant and after 2 min at room tempera-
ture DNA was collected by centrifugation at 15,000g for
1 min at room temperature. DNA was dissolved in 0.3
ml of 10 mM Tris-HCl, 1 mM EDTA, pH 8.0, (TE); 0.15
ml of 7.5 M ammonium acetate added; and DNA precipi-
tated with 2 vol of ethanol at room temperature for 2
min. DNA was again collected by centrifugation in a mi-
crocentrifuge at 15,000g for 1 min at room temperature,
and pellets were washed with 70% ethanol, vacuum
dried, and then resuspended in 0.05 ml of TE. Typically
BACTERIOPHAGE X 231
the yield from this procedure was around 0.8 rg of phage
DNA/l ml of lysate.
2. Small liquid lysates. Starting recombinant phage
stocks and overnight cultures of E. coli strain LE392
were prepared as above. To 50 ~1of an overnight culture
was added 50 ~1 of eluate from a single plaque. The mix-
ture was incubated at 37°C for 10 min to permit phage
adsorption then 5 ml of LB, 10 mM MgS04 was added.
Cultures were shaken at 37°C until lysis was complete
(5-12 h), one drop of chloroform was added to each, and
the lysate was cleared by centrifugation at 12,000g for 10
min at 4°C. To 4 ml of the supernatant was added 2.4 ml
of 20% PEG, 2.5 M NaCl and phage were precipitated on
ice for 30 min. Bacteriophage pellets were collected and
processed for DNA extraction exactly as described for
plate lysates.
Preparation of Bacteriophage DNA from Large Scale
Liquid Lysates
1. Xc18.57. A culture (100 ml) of E. coli strain E514,
which is lysogenic for this temperature-sensitive form of
the virus, was grown in LB at 32°C with shaking to an
A6,,,, of 0.8. The flask was removed to a 45°C water bath
and incubated for 15 min without shaking to effect in-
duction. The culture was then removed to a 38°C water
bath where it was incubated with vigorous shaking for a
further 90 min. The culture was centrifuged at 3000g for
10 min at room temperature and the resultant bacterial
pellet resuspended in 20 ml of PSB. NaCl was added to
the supernatant to a final concentration of 0.9 M fol-
lowed by chloroform (0.1 ml) to initiate bacterial lysis.
The tube was gently rocked at 4°C until lysis was com-
plete (l-2 h). Cell debris was removed by centrifugation
at 12,000g for 10 min at 4°C. PEG was dissolved in the
resultant supernatant to a final concentration of 7.5%
and phage were precipitated on ice for 30 min.
Precipitated bacteriophage were collected by centrifu-
gation at 12,000g for 10 min at 4°C. The supernatant
was discarded and the tube recentrifuged for 1 min to
facilitate removal of any remaining PEG containing su-
pernatant. The precipitate was resuspended in 2.5 ml of
PSB and transferred to a sterile disposable Falcon 15-
ml centrifuge tube; RNaseA and DNaseI were added to
final concentrations of 20 and 5 pg/ml, respectively; and
the tube was incubated at 37°C for 30 min. After
nuclease digestion 2.5 ml of 0.3 M Tris-HCl, 100 mM
EDTA, 1.25% SDS, pH 9.0, was added and the tube incu-
bated a further 10 min at 65°C. Ice-cold 3 M potassium
acetate, pH 4.8 (2.5 ml), was added and the tube incu-
bated on ice for 10 min. Insoluble material was removed
by centrifugation at 12,000g for 10 min at 4°C and most
of the supernatant (7.0 ml) removed taking care to ex-
clude any flocculent insoluble material. Isopropanol(5.0
ml) was mixed with the supernatant and after 2 min at
232 TREVOR J. LOCKETT

room temperature DNA was recovered by centrifugation A B C D E

at 12,000g for 5 min at room temperature. DNA was dis-
solved in 0.6 ml of 10 mM Tris-HCl, 1 mni EDTA, pH 1,2 1,2 1,2 1,2 1,2
8.0 (TE); 0.3 ml transferred to each of two 1.5-ml micro-
centrifuge tubes; 0.15 ml of 7.5 M ammonium acetate
added to each tube; and DNA precipitated with 2 vol of
ethanol at room temperature for 2 min. DNA was col-
lected by centrifugation at 15,000g for 1 min at room
temperature, and pellets were washed with 70% ethanol,
vacuum dried, and then resuspended in 0.15 ml of TE
per tube.
2. Recombinant X clones. Starting high titer phage
stocks were derived from plate lysates prepared from the FIG. 1. EcoRl digests of SDS/potassium acetate and commercial
original recombinant plaque as described above. A cul- Xc1857 DNA in varying concentrations of spermidine. About 0.15 pg
of SDS/potassium acetate prepared (lane 1) or commercial (lane 2)
ture of E. coli strain LE392 was grown overnight in LB, DNA was digested with EcoRl in the presence of 4 mM (A), 2 mM
0.1% maltose with shaking and 1.0 ml of this culture was (B), 1 mM (C), 0.5 mM (D), or 0 mM (E) spermidine. Samples were
added to approximately 5 X lo6 phage in 1.0 ml of PSB. electrophoresed in a 0.8% agarose gel which was stained with ethidium
The mixture was incubated at 37°C for 10 min to permit bromide.
phage adsorption then added to 100 ml of LB, 10 mM
MgS04. Cultures were shaken at 37°C until lysis was
complete (5-12 h). Chloroform (0.25 ml) was added to
(e.g., labeling of DNA with polynucleotide kinase and
each lysate and NaCl added to a concentration of 0.9 M.
DNA ligation) is known to be improved by the addition
The lysates were chilled on ice for 10 min and then
of spermidine (5). In addition spermidine (3-5 mM) has
cleared by centrifugation at 10,OOOg for 10 min at 4°C.
been demonstrated to restore restrictability of DNA
Solid PEG (7.5g) was dissolved in each supernatant and
fragments isolated from agarose gels (9). Spermidine
phage were precipitated on ice for 30 min.
was therefore added to restriction endonuclease diges-
Collection of precipitated bacteriophage and extrac-
tions of SDS/potassium acetate isolated X DNA at a fi-
tion of DNA were carried out exactly as described above
nal concentration of 4 mM. Figure 1A indicates that this
for Xc1857.
addition successfully overcame the block to digestion of
3. Ammonium sulfate method. As described in (7). DNA prepared by this method. More detailed analysis
All experiments involving recombinant molecules revealed that concentrations of spermidine as low as 0.5
were carried out in accordance with the guidelines pub- mM were equally effective at restoring restrictability
lished by the Australian Genetic Manipulations Advi- (Figs. lB-1D). Thus the standard restriction digestion
sory Committee. condition applied to X DNA prepared by this method was
modified to include spermidine at a final concentration
Restriction Endonuclease Digestion and of 1 mM.
Gel Electrophoresis The procedure described for the purification of DNA
Approximately 0.15 pg of DNA was digested with a from PEG-precipitated bacteriophage was based on a
fourfold excess of enzyme for 2 h at 37°C in a final reac- method commonly used for the small scale isolation of
tion volume of 6 ~1. Except where otherwise indicated, 1 nucleic acids from the fruit fly Drosophila melanogaster
mM spermidine was included to supplement the man- (10,ll). Thus in its original form this method included
ufacturers recommended conditions. Samples were the addition of diethyl pyrocarbonate to inactivate
heated at 65°C and loaded directly on 0.8% agarose gels nucleases immediately prior to the heating step (see Ma-
prepared in TBE buffer (5). After electrophoresis gels terials and Methods) and a chloroform extraction after
were stained with ethidium bromide. the potassium acetate precipitation step to further pu-
rify the nucleic acids. To determine whether either of
these steps was essential, DNA was prepared from three
RESULTS
independent small scale liquid lysate preparations incor-
This SDS/potassium acetate procedure produces X porating chloroform extraction alone, diethyl pyrocar-
DNA in high yield which shows minimal contamination bonate and chloroform treatments, or neither treatment
by RNA or chromosomal DNA. However, this DNA (Fig. 2A, lanes 1,2, and 3, respectively). The figure shows
proved difficult to digest with restriction endonucleases that neither step is necessary for the preparation of
under standard conditions (Fig. 1E). The efficiency of a DNA suitable for restriction analysis from PEG-precipi-
number of enzyme-mediated DNA modifying reactions tated bacteriophage h.
 DNA PURIFICATION FROM BACTERIOPHAGE X 233

The SDS/potassium acetate method is equally appli- A 6 C D

cable to the preparation of DNA from phage stocks pre-
pared from plate lysates. The same recombinant phage 1,2,3 1,2,3 1,2,3 1,2,3
stock used to prepare the liquid lysate shown in Fig. 2A
was used to prepare lysates from single LB, 10 mM
MgS04 agarose, or agar plates (Fig. 2, lanes 2 and 3, re-
spectively). The figure shows that lysates prepared from
either agarose or agar plates give rise to DNA which can
be cut by EcoRI. In each case complete digestion of the
phage DNA was independent of chloroform extraction.
The quality of the X DNA resulting from this isolation
procedure was compared with that of similar DNA ob-
tained commercially or prepared by the ammonium sul-
fate method by carrying out restriction digestions using FIG. 3. Comparison by limit digestion with EcoRl of X DNAs pre-
decreasing ratios of enzyme to DNA. Figure 3 indicates pared by different methods. Ad857 DNA prepared by the SDS/potas-
sium acetate method (lane l), prepared by the ammonium sulfate
that all three DNA samples begin to show approximately method (lane 2), or obtained commercially (lane 3) was digested with
equal levels of partial digestion when the ratio of enzyme EcoRl at enzyme to DNA ratios of 4 units/pg (A), 2 units/pg (B), 1
to DNA is reduced to 1 unit/pg in a 60-min reaction. By unit/pg (C), and 0.5 unit/pg (D) for 1 h at 3’7°C. Samples were electro-
this criterion of restrictability in the presence of spermi- phoresed in a 0.8% agarose gel which was stained with ethidium bro-
mide.
dine, X DNA prepared by the SDS/potassium acetate
method appears to be of equal quality to that obtained
either commercially or by the ammonium sulfate proce- been used successfully to transform competent E. coli
dure. Since the SDS/potassium acetate preparation of strain LE392 (data not shown).
Xc1857 DNA shown in Fig. 3 had been stored at 4°C for
over 12 months at the time of test, it is apparent that DISCUSSION
DNA prepared in this way is also quite stable.
DNA prepared using this SDS/potassium acetate The X DNA purification method presented here com-
method was cut by all restriction endonucleases tested bines classical PEG purification of phage particles (4,5)
(BamHl, Hindlll, Sail, Xbal, and EcoRl) and the re- with a modification of the SDS/potassium acetate
sultant fragments were readily subcloned. The DNA method for DNA purification often used for the small
could be labeled efficiently by nick-translation and it has scale extraction of nucleic acids from D. mehogaster
(10,ll). The original SDS/potassium acetate procedure
as applied to fly DNA purification and that previously
described for preparation of h DNA from plate lysates
A B C (8) both used diethylpyrocarbonate to inactivate
nucleases. In addition a chloroform extraction was in-
1,2,3 1,2 1,2
cluded in the fly DNA preparation protocol to further
purify the nucleic acids. Neither of these steps proved to
be necessary for DNA isolation from PEG-precipitated
bacteriophage. Contamination by chromosomal DNA or
RNA is undetectable in these preparations and DNA is
recovered in high yield. Although spermidine must be
included in all restriction endonuclease digestions, stan-
dard reaction conditions can be used for all other com-
mon molecular biological procedures. The mechanism
by which spermidine facilitates restriction endonuclease
digestion is not clear but is likely to involve the strong
FIG. 2. Effect of X DNA extraction modifications and lysate source
on EcoRl digestion. Phage stocks from a recombinant clone in the X interactions known to occur between spermidine and
EMBL3 vector were prepared from miniliquid lysates (A), agarose DNA. One possibility might be that spermidine dis-
plate lysates (B), or agar plate lysates (C). DNAs were prepared from places from the DNA some inhibitory component of the
these without either diethyl pyrocarbonate treatment or chloroform mixture so rendering the nucleic acid accessible to re-
extraction (lane l), without diethyl pyrocarbonate treatment but with striction enzymes. Such an inhibitory component is un-
chloroform extraction (lane 2), or with both diethyl pyrocarbonate
treatment and chloroform extraction (A, lane 3). DNAs were digested
likely to be proteinaceous in nature since extraction with
with EcoRl, and samples electrophoresed in a 0.8% agarose gel which phenol and chloroform does not relieve the inhibition
was stained with ethidium bromide. (data not shown).
234 TREVOR J. LOCKETT

The SDS/potassium acetate method for bacterio- ing, a Practical Approach (Glover, D., Ed.), Vol. 1, pp. 49-78, IRL,
Oxford.
phage X DNA purification described here is quick, effi-
cient, and inexpensive being ideally suited to the rapid 4. Yammamoto, K. R., Albert, B., Benzinger, R., and Treiber, G.
(1970) Virology 40.734-744.
preparation of DNA from multiple small lysates.
5. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) in Molecular
Cloning: A Laboratory Manual, pp. 83-85, Cold Spring Harbor
ACKNOWLEDGMENTS Laboratory, Cold Spring Harbor, NY.
The author is grateful to Drs. P. Jennings and P. Malloy for helpful 6. Helms, C., Graham, M. Y., Dutchik, J. E., and Olson, M. V. (1985)
discussions and critical reading of the manuscript and to A. McGill for DNA 4,39-49.
assistance with manuscript preparation. 7. Ziai, M. R., Giordano, A., Armandola, E., and Ferrone, S. (1988)
Anal. Biochem. 171,192-196.
8. Davis, R. W., Botstein, D., and Roth, J. R. (1986) Aduanced Bacte-
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2. Young, R. A., and Davis, R. W. (1983) Proc. Natl. Acad. Sci. USA 433.
80,1194-1198. 11. Jowett, T. (1986) in Drosophila, a Practical Approach (Roberts,
3. Huynh, T. V., Young, R. A., and Davis, R. W. (1985) in DNA Clon- D. B., Ed.), pp. 275-286, IRL, Oxford.