ANALYTICAL BIOCHEMISTRY 175,196-20 1 ( 1988)

Filter-Supported Preparation of X Phage DNA

R. JAKOBI, S. WIEMANN, AND W. PYERIN'

Institute ofExperimental Pathology, German Cancer Research Center, im Neuenheimer Feld 280,
D-6900 Heidelberg, Federal Republic of Germany
Received April 1, 1988

A rapid and simple method is described for the isolation of DNA from phage X which requires
neither special equipment nor expensive material such as cesium chloride for ultracentrifugation
nor extractions with organic solvents or ethanol precipitation. Microgram quantities of A DNA
are obtained in less than 2 h from 90-mm plate lysates or 5-ml liquid cultures. The method
allows the simultaneous isolation of large numbers of probes, e.g., clones from phage libraries.
X phages are precipitated by polyethylene glycol/sodium chloride and recovered by low speed
centrifugation onto glass fiber filters positioned in disposable syringes. The DNA of phages is
released by a 50% formamide/ M sodium perchlorate solution, washed in filter-bound form,
eluted with a small volume of low-salt buffer or water, and finally recovered by centrifugation.
Comparison of the DNA isolated by this method with that obtained by two conventional proce-
dures reveals both a similar recovery and a similar suitability for restriction enzyme digestion
and subcloning. D 1988 Academic Res, Inc.
KEY WORDS: phage lambda; DNA isolation; molecular cloning.

The Escherichia coli phage X has become solvents are necessary, the DNA of large
one of the most frequently applied cloning numbers of clones may be prepared in paral-
vectors in construction of genomic or cDNA lel leading to a considerable speeding-up of
libraries for reasons such as the ability to ac- the procedure. A 5-ml liquid culture or a 90-
comodate larger inserts than plasmid vectors mm plate lysate was enough to yield DNA in
or having higher transfer frequencies. The microgram quantities, i.e., amounts suitable
screening of X phage libraries for selected for several restriction digests. The DNA pre-
DNA fragments makes it necessary to isolate pared by this method can also be used for pre-
DNA in microgram quantities from a large parative purposes such as subcloning.
number of clones. The standard methods are
either reliable but slow (l-3) or fast but not MATERIALS AND METHODS
reliable for preparative use (1,4). Steps like
The bacterial strains E. coli C600 and E.
cesium chloride gradient centrifugations or
coli C600hflA, the phage vector Xgt 10 and the
repeated extractions with phenol and chloro-
in vitro-packaging extracts were purchased
form are very time-consuming if DNA from
from Promega Biotec. Xgt lOMC1 is a con-
larger numbers of clones has to be prepared.
struct of Xgt 10 and a 604-bp cDNA insert (5)
Here we describe a method for the simulta-
cloned in the EcoRI site. Restriction enzyme
neous isolation of X phage DNA from differ-
EcoRI was purchased in a highly concen-
ent clones. The method utilizes the property trated batch from Boehringer-Mannheim, T4
of DNA to bind to glass fiber filters as the sep- DNA-ligase from New England Biolabs, low
aration principle. Because neither gradient melting agarose Nusieve GTG from Biozym
centrifugations nor extractions with organic Diagnostik, 3250 Hameln 1, and DEAE cel-
lulose DE 52 and GF/C glass fiber filters from
’ To whom all correspondence should be addressed. Whatman. DEAE cellulose was equlibrated

0003-2697188 $3.00 196

Copyright 0 1988 by Academic Press, Inc.
All rights of reproduction in any form reserved.
 PREPARATION OF X PHAGE DNA 197

as described (4). The buffers used were TE ( 10 tated by adding polyethylene glycol and so-
mM Tris-HCi, pH 8, 1 mM Na,EDTA) and dium chloride to final concentrations of 7
TAE (40 mM Tris, 40 mM acetic acid, 2 mM and 2%, respectively. The phages are immo-
NazEDTA). bilized on GF/C glass fiber filters positioned
Phages were grown as liquid cultures or at the bottom of a 5-ml disposable syringe by
plate lysates as described (1,3,4). Standard applying the phage precipitate to the syringe
preparations of phage DNA were performed, and centrifuging at 40g for 5 min. The DNA
proceeding from 40-ml liquid cultures, either of phages is then released by centrifuging 1 ml
according to (2) by RNase A and DNase I di- of 4 M sodium perchlorate/50% formamide
gestion of the clear phage supernatant, poly- solution through the filter as before. In the
ethylene glycol/sodium chloride precipita- same manner, the filter-bound DNA is de-
tion of phages followed by centrifugation, proteinized by 1 ml of 4 M sodium perchlo-
SDS’ lysis, several phenol extractions, and rate and desalted by 1 ml of 70% ethanol. Af-
ethanol precipitation or according to ( 1,3) by ter air-drying for 5 min, the DNA is eluted
pelleting of phages through ultracentrifuga- from the filters with 50- 100 ~1 0.1 X TE by
tion of the clear phage supernatant followed incubating at 60-65°C for 15-30 min and
by a two-step cesium chloride gradient, for- vortexing for a few seconds. This may be car-
mamide treatment, and ethanol precipita- ried out within the syringe or after the filters
tion. Restriction enzyme digestions, gel elec- are placed in 0.5-ml tubes. The DNA solution
trophoresis, insert isolation with Nusieve low is then centrifuged into 1.5-ml tubes either
melting agarose, ligation, transformation, through the outlet of the syringe at 200g for
and in vitro-packaging were performed ac- 3-5 min or through a hole punched into the
cording to the customers information. Prepa- bottom of the 0.5-ml tubes at 10,OOOg for
ration of replicas and hybridization were car- 30 s.
ried out as described (6) with the exception
that a biotinylated probe and nylon mem- Results
branes were used. Densitometric scanning of Removal of Bacterial Nucleic Acids and Agar
photographic negatives of gels was carried out Contaminants
in a LKB Ultrascan XL laser densitometer
with 2400 gelscan XL software. The phages (Xgtl OMCI) obtained from 5-
The recommended procedure for the filter- ml liquid cultures or 90-mm plate lysates
supported preparation of X phage DNA, the were separated from bacterial debris by cen-
development of which is described in this pa- trifugation and from bacterial nucleic acids as
per, is the following. X phages obtained from well as negatively charged agar contaminants
liquid cultures or plate lysates are separated by the DEAE cellulose procedure (4) either
from bacterial debris by centrifugation at batch-wise or via column chromatography
13,000g for 10 min. To remove bacterial (Fig. 1). Importantly, while DNA obtained
nucleic acids and agar contaminants, the su- from phages grown in liquid cultures was di-
pernatant is incubated under shaking at room gested with EcoRI both with and without
temperature for 5 min with 1 vol of DEAE DEAE treatment (lane 2 vs lane 4), DNA
cellulose equilibrated in LB-medium fol- from plate lysates was digested only after re-
lowed by centrifugation at 2,500g for 15 min. moval of negatively charged agar contami-
(Alternatively, the equilibrated DEAE cellu- nants by DEAE cellulose treatment (lane 6 vs
lose may be filled into a column and the su- lane 8).
pernatant applied). The phages are precipi-
Precipitation of Phages
2 Abbreviations used: SDS, sodium dodecyl sulfate; To precipitate the phages, polyethylene
RCF, relative centrifugal force. glycol and sodium chloride were added to the
198 JAKOBI, WIEMANN, AND PYERIN

effect on the recovery. Therefore, polyethyl-

ene glycol/sodium chloride-treated phage so-
lutions were applied immediately to the next
Kb step, the immobilization of phages on glass
23.1- fiber filters.
9.4-
4.4-
Immobilization of Phageson GlassFiber
2.3- Filters and Washing of Released Filter-
Bound Phage DNA
1.1. A precut piece of a GF/C glass fiber filter
was put onto the bottom of a 5-ml disposable
0.6- syringe and the syringe inserted into an ap-
0.3- propriate centrifuge tube. Then, the phage
precipitate was applied and freed from the su-
pernatant by centrifugation at 4Og, usually
FIG. I. Agarose gel electrophoresis of DNA isolated requiring 5 min. Shortening of centrifugation
from phage solutions with and without DEAE cellulose time was found possible by increasing the
treatment. DNA was isolated by the filter-supported pro- RCF up to about 150g. At this RCF, how-
cedure as given under Materials and Methods from ever, damage to the filter may occur. In order
XgtlOMCI grown in 5-ml liquid cultures (lanes 1-4) or
obtained from 90-mm plate lysates (lanes 5-8) with to release the DNA from phages, 1 ml of a 4 M
DEAE cellulose treatment (lanes 3,4 and 7,8) and with- sodium perchlorate/50% formamide solution
out (lanes 1, 2 and 5,6). Aliquots of undigested (lanes 1, was applied to the filter, followed by centrifu-
3, 5, 7) and EcoRI digested (lanes 2, 4, 6, 8) XgtlOMCI gation as above. Under this condition, the
DNA were applied to a 1% agarose gel followed by elec- DNA immediately bound to the glass fiber
trophoresis in TAE buffer. Figures indicate positions of
molecular weight markers (X Hind111 and Phi X 174
HaeIII).

DEAE-purified phage solution. Since pub-

lished methods vary considerably in concen- l-
trations specified, we tested concentration
ranges for optimal recovery. Polyethylene
glycol concentrations of 5% and above were
found to precipitate roughly 80-90% of the O.S-
phages; recoveries dramatically dropped at
polyethylene concentrations below 5% (Fig.
2). Addition of sodium chloride (tested up to
5% in presence of 12% polyethylene glycol)
had little effect on the recovery. Apparently,
OI 0 2 4 6 6 10 12

the amount of sodium chloride present in the PEG [z]

LB-medium or the X dilution buffer was al- FIG. 2. Dependance of phage precipitation on polyeth-
ready sufficient. ylene glycol concentration. Given are the amounts of
Standard phage precipitation protocols DNA obtained from phage precipitates at the shown con-
with polyethylene glycol/sodium chloride in- centrations of polyethylene glycol in the presenceof 5%
sodium chloride. The DNA was quantified by agarose gel
clude incubation on ice for 1 h. To speed up electrophoresis followed by densitometric scanning of
the procedure, shorter incubation times on photographic negatives of gels; 2.5 X 10” phages from
ice were tested and, in addition, freezing to 2.5-ml liquid culture were employed with a theoretical
-20°C. Neither effort had any significant recovery of 1.2 pg DNA.
 PREPARATION OF X PHAGE DNA 199

filter upon release. If sodium perchlorate was

omitted, the recovery dropped dramatically.
The filter-immobilized DNA was deprotein-
ized by adding 1 ml of 4 M sodium perchlo-
rate solution and centrifuging as before. Fi- Kb
nally, salts were washed out with 1 ml of 70% 23.1-
ethanol in the same way. The latter wash was 9.4-
found sufficient in a test series including ab- 4.4-
solute ethanol, 70% ethanol, acetone, chloro-
form, and several combinations of these. The 2.3-
filters were freed from ethanol by allowing
them to air-dry for 15 min within the syringe 1.1.
or 5 min outside it.
0.69
El&ion of the Filter-Bound Phage DNA
The filter-bound phage DNA could be 0.3-
eluted with low salt buffers such as 0.1 X TE
or simply with water, the fragment size of
DNA to be eluted determining the volume.
FIG. 3. Recovery of X phage DNA upon application of
Short DNA fragments require small elution the filter-supported method in comparison to that of two
volumes, i.e., lo-20 ~1(7-lo), while elution conventional methods. DNA from XgtlOMCI phages
of longer fragments such as the 40 to 50-kb X grown in 40-ml liquid cultures were isolated by cesium
DNA demands larger volumes. The best re- chloride gradient centrifugation followed by formamide
sults were obtained with 50-100 ~10.1 X TE. extraction (lane l), by SDS lysis of phages followed by
phenol extractions (lane 2), and by the new filter-sup-
Recovery was increased by incubation for ported method (lane 3). Two probes each were prepared.
15-30 min at 60-65°C and vortexing for a In case of SDS lysis/phenol extraction (lane 2), RNase
few seconds. The eluted DNA was centri- was added to the EcoRI restriction digest to remove
fuged through the filter at 200ginto an appro- RNA. The isolates were applied onto a 1% agarose gel
priate tube positioned at the syringe outlet. and electrophorized in TAE buffer. Figures indicate posi-
tions of molecular weight markers (X Hind111 and Phi
During incubation and vortexing, the outlet X 174 HueIII).
of the syringe was stoppered. Alternatively,
the filters were transfered to small tubes (usu-
ally 0.5-ml tubes) for elution. In this case, the with that of conventional methods, X phage
tube bottoms were perforated after incuba- DNA was isolated from liquid cultures both
tion and vortexing and the eluted DNA was
by a two-step cesium chloride gradient puri-
centrifuged directly into another test tube
fication followed by formamide extraction of
placed underneath. Elution outside the sy- the phage DNA (1,3) and by a SDS-lysis
ringe in small tubes is the better method. In- method of phages with subsequent phenol ex-
creasing the elution volume to more than 100 tractions of the released DNA (2). The DNA
~1 per 5-ml liquid culture or 90-mm plate ly- prepared by all three methods is similar; i.e.,
sate should be avoided because the DNA then upon agarose gel electrophoresis the XgtlO
has to be concentrated by ethanol precipita-
arms at 32,7 10 and at 10,630 bp as well as
tion.
the 604-bp MCI insert are discernible in each
Comparison of DNA Obtained by the Filter- case (Fig. 3). The recovery was comparable
Supported Preparation and by with all three methods, i.e., more than 80%,
Conventional Methods while the purity differed somewhat. Purity
In order to compare the recovery of DNA was best with the cesium cholide gradient
by our filter-supported preparation method method (lane 1). DNA isolated by the
200 JAKOBI, WIEMANN. AND PYERIN

SDS-lysis method (lane 2) contained large in addition contaminants originating from

amounts of RNA which had to be removed the agar growth medium which considerably
by adding RNase to restriction digests. The inhibit many of the DNA-modifying en-
DNA isolated by the new filter-supported zymes hindering preparative usage of the
method was not totally pure (lane 3). How- phage DNA. The DEAE cellulose treatment
ever, it was pure enough to be as successfully therefore offers the advantage of working also
used for preparative purposes as the DNA ob- with plate lysates providing higher phage ti-
tained by the other two methods. ters than liquid cultures.
After the bacterial nucleic acids are re-
Preparative Usage of Isolated DNA: moved, the phages are precipitated. Precipi-
Recloning of Insert DNA tation by acetic acid as described for phage M
13 (7) or with ethanol and acetone resulted in
For subcloning, Xgt 1OMCI DNA obtained
very low recoveries. We therefore returned to
by the filter-supported method was digested
the conventional polyethylene precipitation
with EcoRI, the restriction fragments were
procedure and found it optimal in recovery
separated by electrophoresis in low melting
and speed. At final concentrations of 5%
agarose, and the 604-bp MCI band was cut
polyethylene glycol and above, recoveries of
out. The gel slice was melted at 68°C and
phage DNA have been better than 80% and
cooled to 37°C MCI DNA religated into de-
none of the time-consuming incubations on
phosphorylated Xgt 10 DNA, and the phages
ice or at temperatures below the freezing
plated out on E. coli C600. To ensure that
point are necessary. The precipitated phages
inserted DNA was really MCI DNA and not
can rather immediately be immobilized on
any contaminating bacterial DNA of compa-
glass fiber filters, in which state the release of
rable size, a nylon replica from the plate was DNA is accomplished.
hybridized to a biotinylated plasmid contain-
Formamide treatment of phage X appears
ing the MCI insert. Only the clear plaque-
to cause the DNA in the phage to be ejected
forming units exhibiting positive signals con-
from the tail (1). On the other hand, DNA has
tained an insert as proved by concomitant
been shown to bind to glass fiber filters in the
DNA isolation and restriction analysis (data
presence of high concentrations of sodium
not shown). Also, transformation of plasmid perchlorate (7- 10). Combining these obser-
vectors with a subcloned insert (ligated as vations, the immobilized phages were treated
above) was found possible. This makes un-
by 50% formamidef4 M sodium perchlorate
ambiguously clear that the filter-supported
solution and found to dissociate phage DNA
DNA preparation is a reliable method for
from phage protein, the DNA being immedi-
preparative purposes.
ately bound to the filters upon release,
whereas proteins are unable to bind under
DISCUSSION
such conditions. This makes it possible to de-
The separation of bacterial DNA from the proteinize and desalt the DNA conveniently
phage DNA can conveniently be performed and quickly. Deproteinization is achieved by
at a stage where the phages are intact. At this adding 4 M sodium perchlorate as washing so-
stage, bacterial DNA is digestable selectively lution to the filter-bound DNA followed by a
with DNase. The DNase, however, has to be short low-speed centrifugation. The depro-
removed afterward by phenol extraction (2). teinization in solution with organic solvents
To avoid working with this poisonous and such as phenols or chloroform (2) with all
caustic material (11) and to shorten working their undesirable implications if inhaled are
time, we attempted to remove the bacterial omitted as is the rather expensive cesium
nucleic acids chromatographically by the chloride gradient ultracentrifugation ( 1,3).
DEAE cellulose procedure (4). This removed Desalting of the filter-bound DNA is carried
 PREPARATION OF X PHAGE DNA 201

out similarly. A short wash with 70% ethanol ACKNOWLEDGMENTS

is sufficient. Ethanol, a known inhibitor of
We are grateful to Prof. V. Kinzel for his continuous
enzymes for the further processing of DNA, encouragement and support, Dr. J. Reed for semantic
is removed quantitatively from the filters assistance, and M. Kaszkin for help in computer graph-
simply by evaporation and checking by ics. This work was supported in part by the Sonderver-
smelling. Desalting and concentration by eth- mijgen des Deutschen Krebsforschungszentrums and the
anol precipitation, a necessary step in con- Deutsche Forschungsgemeinschat?.
ventional procedures ( l-4), becomes super-
fluous. In fact, the possibility of carrying out REFERENCES
all the necessary washing steps so easily,
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Advanced Bacterial Genetics: A Manual for Ge-
major advantage of this method. Since the netic Engineering, Cold Spring Harbor Labora-
elution of DNA from the filters is again easily tory, Cold Spring Harbor, NY.
accomplished by adding a small volume of 2. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982)
solution or of water followed by a short incu- Molecular Cloning: A Laboratory Manual, Cold
bation and low-speed centrifugation, the Spring Harbor Laboratory. Cold Spring Harbor,
NY.
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3. Glover, D. M. ( 1985) DNA Cloning, Vol. 1, IRL
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was, without exception, suitable for restric- 6. HamesB. D., and Higgins, S. J. (Eds.) (1985) Nucleic
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