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
one of the most frequently applied cloning
vectors in construction of genomic or cDNA
libraries for reasons such as the ability to ac-
comodate larger inserts than plasmid vectors
or having higher transfer frequencies. The
screening of X phage libraries for selected
DNA fragments makes it necessary to isolate
DNA in microgram quantities from a large
number of clones. The standard methods are
either reliable but slow (l-3) or fast but not
reliable for preparative use (1,4). Steps like
cesium chloride gradient centrifugations or
repeated extractions with phenol and chloro-
form are very time-consuming if DNA from
larger numbers of clones has to be prepared.
Here we describe a method for the simulta-
neous isolation of X phage DNA from differ-
ent clones. The method utilizes the property
of DNA to bind to glass fiber filters as the sep-
aration principle. Because neither gradient
centrifugations nor extractions with organic
’ To whom all correspondence should be addressed.
solvents are necessary, the DNA of large
numbers of clones may be prepared in paral-
lel leading to a considerable speeding-up of
the procedure. A 5-ml liquid culture or a 90-
mm plate lysate was enough to yield DNA in
microgram quantities, i.e., amounts suitable
for several restriction digests. The DNA pre-
pared by this method can also be used for pre-
parative purposes such as subcloning.
MATERIALS AND METHODS
The bacterial strains E. coli C600 and E.
coli C600hflA, the phage vector Xgt 10 and the
in vitro-packaging extracts were purchased
from Promega Biotec. Xgt lOMC1 is a con-
struct of Xgt 10 and a 604-bp cDNA insert (5)
cloned in the EcoRI site. Restriction enzyme
EcoRI was purchased in a highly concen-
trated batch from Boehringer-Mannheim, T4
DNA-ligase from New England Biolabs, low
melting agarose Nusieve GTG from Biozym
Diagnostik, 3250 Hameln 1, and DEAE cel-
lulose DE 52 and GF/C glass fiber filters from
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
as described (4). The buffers used were TE ( 10
mM Tris-HCi, pH 8, 1 mM Na,EDTA) and
TAE (40 mM Tris, 40 mM acetic acid, 2 mM
NazEDTA).
Phages were grown as liquid cultures or
plate lysates as described (1,3,4). Standard
preparations of phage DNA were performed,
proceeding from 40-ml liquid cultures, either
according to (2) by RNase A and DNase I di-
gestion of the clear phage supernatant, poly-
ethylene glycol/sodium chloride precipita-
tion of phages followed by centrifugation,
SDS’ lysis, several phenol extractions, and
ethanol precipitation or according to ( 1,3) by
pelleting of phages through ultracentrifuga-
tion of the clear phage supernatant followed
by a two-step cesium chloride gradient, for-
mamide treatment, and ethanol precipita-
tion. Restriction enzyme digestions, gel elec-
trophoresis, insert isolation with Nusieve low
melting agarose, ligation, transformation,
and in vitro-packaging were performed ac-
cording to the customers information. Prepa-
ration of replicas and hybridization were car-
ried out as described (6) with the exception
that a biotinylated probe and nylon mem-
branes were used. Densitometric scanning of
photographic negatives of gels was carried out
in a LKB Ultrascan XL laser densitometer
with 2400 gelscan XL software.
The recommended procedure for the filter-
supported preparation of X phage DNA, the
development of which is described in this pa-
per, is the following. X phages obtained from
liquid cultures or plate lysates are separated
from bacterial debris by centrifugation at
13,000g for 10 min. To remove bacterial
nucleic acids and agar contaminants, the su-
pernatant is incubated under shaking at room
temperature for 5 min with 1 vol of DEAE
cellulose equilibrated in LB-medium fol-
lowed by centrifugation at 2,500g for 15 min.
(Alternatively, the equilibrated DEAE cellu-
lose may be filled into a column and the su-
pernatant applied). The phages are precipi-
2 Abbreviations used: SDS, sodium dodecyl sulfate;
RCF, relative centrifugal force.
OF X PHAGE DNA 197
tated by adding polyethylene glycol and so-
dium chloride to final concentrations of 7
and 2%, respectively. The phages are immo-
bilized on GF/C glass fiber filters positioned
at the bottom of a 5-ml disposable syringe by
applying the phage precipitate to the syringe
and centrifuging at 40g for 5 min. The DNA
of phages is then released by centrifuging 1 ml
of 4 M sodium perchlorate/50% formamide
solution through the filter as before. In the
same manner, the filter-bound DNA is de-
proteinized by 1 ml of 4 M sodium perchlo-
rate and desalted by 1 ml of 70% ethanol. Af-
ter air-drying for 5 min, the DNA is eluted
from the filters with 50- 100 ~1 0.1 X TE by
incubating at 60-65°C for 15-30 min and
vortexing for a few seconds. This may be car-
ried out within the syringe or after the filters
are placed in 0.5-ml tubes. The DNA solution
is then centrifuged into 1.5-ml tubes either
through the outlet of the syringe at 200g for
3-5 min or through a hole punched into the
bottom of the 0.5-ml tubes at 10,OOOg for
30 s.
Results
Removal of Bacterial Nucleic Acids and Agar
Contaminants
The phages (Xgtl OMCI) obtained from 5-
ml liquid cultures or 90-mm plate lysates
were separated from bacterial debris by cen-
trifugation and from bacterial nucleic acids as
well as negatively charged agar contaminants
by the DEAE cellulose procedure (4) either
batch-wise or via column chromatography
(Fig. 1). Importantly, while DNA obtained
from phages grown in liquid cultures was di-
gested with EcoRI both with and without
DEAE treatment (lane 2 vs lane 4), DNA
from plate lysates was digested only after re-
moval of negatively charged agar contami-
nants by DEAE cellulose treatment (lane 6 vs
lane 8).
Precipitation of Phages
To precipitate the phages, polyethylene
glycol and sodium chloride were added to the
198 JAKOBI, WIEMANN,
Kb
23.1-
9.4-
4.4-
2.3-
1.1.
0.6-
0.3-
FIG. I. Agarose gel electrophoresis of DNA isolated
from phage solutions with and without DEAE cellulose
treatment. DNA was isolated by the filter-supported pro-
cedure as given under Materials and Methods from
XgtlOMCI grown in 5-ml liquid cultures (lanes 1-4) or
obtained from 90-mm plate lysates (lanes 5-8) with
DEAE cellulose treatment (lanes 3,4 and 7,8) and with-
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
DNA were applied to a 1% agarose gel followed by elec- DNA immediately
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-
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
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,
the amount of sodium chloride present in the
LB-medium or the X dilution buffer was al-
ready sufficient.
Standard phage precipitation protocols
with polyethylene glycol/sodium chloride in-
clude incubation on ice for 1 h. To speed up
the procedure, shorter incubation times on
ice were tested and, in addition, freezing to
-20°C. Neither effort had any significant
AND PYERIN
effect on the recovery. Therefore, polyethyl-
ene glycol/sodium chloride-treated phage so-
lutions were applied immediately to the next
step, the immobilization of phages on glass
fiber filters.
Immobilization of Phageson GlassFiber
Filters and Washing of Released Filter-
Bound Phage DNA
A precut piece of a GF/C glass fiber filter
was put onto the bottom of a 5-ml disposable
syringe and the syringe inserted into an ap-
propriate centrifuge tube. Then, the phage
precipitate was applied and freed from the su-
pernatant by centrifugation at 4Og, usually
requiring 5 min. Shortening of centrifugation
time was found possible by increasing the
RCF up to about 150g. At this RCF, how-
ever, damage to the filter may occur. In order
to release the DNA from phages, 1 ml of a 4 M
sodium perchlorate/50% formamide solution
gation as above. Under this condition, the
bound to the glass fiber
l-
O.S-
OI 0 2 4 6 6 10 12
PEG [z]
FIG. 2. Dependance of phage precipitation on polyeth-
ylene glycol concentration. Given are the amounts of
DNA obtained from phage precipitates at the shown con-
centrations of polyethylene glycol in the presenceof 5%
sodium chloride. The DNA was quantified by agarose gel
electrophoresis followed by densitometric scanning of
photographic negatives of gels; 2.5 X 10” phages from
2.5-ml liquid culture were employed with a theoretical
recovery of 1.2 pg DNA.
 PREPARATION
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-
nally, salts were washed out with 1 ml of 70%
ethanol in the same way. The latter wash was
found sufficient in a test series including ab-
solute ethanol, 70% ethanol, acetone, chloro-
form, and several combinations of these. The
filters were freed from ethanol by allowing
them to air-dry for 15 min within the syringe
or 5 min outside it.
El&ion of the Filter-Bound Phage DNA
The filter-bound phage DNA could be
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.
Short DNA fragments require small elution
volumes, i.e., lo-20 ~1(7-lo), while elution
of longer fragments such as the 40 to 50-kb X
DNA demands larger volumes. The best re-
sults were obtained with 50-100 ~10.1 X TE.
Recovery was increased by incubation for
15-30 min at 60-65°C and vortexing for a
few seconds. The eluted DNA was centri-
fuged through the filter at 200ginto an appro-
priate tube positioned at the syringe outlet.
During incubation and vortexing, the outlet
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
tube bottoms were perforated after incuba-
tion and vortexing and the eluted DNA was
centrifuged directly into another test tube
placed underneath. Elution outside the sy-
ringe in small tubes is the better method. In-
creasing the elution volume to more than 100
~1 per 5-ml liquid culture or 90-mm plate ly-
sate should be avoided because the DNA then
has to be concentrated by ethanol precipita-
tion.
Comparison of DNA Obtained by the Filter-
Supported Preparation and by
Conventional Methods
In order to compare the recovery of DNA
by our filter-supported preparation method
OF X PHAGE DNA 199
Kb
23.1-
9.4-
4.4-
2.3-
1.1.
0.69
0.3-
FIG. 3. Recovery of X phage DNA upon application of
the filter-supported method in comparison to that of two
conventional methods. DNA from XgtlOMCI phages
grown in 40-ml liquid cultures were isolated by cesium
chloride gradient centrifugation followed by formamide
extraction (lane l), by SDS lysis of phages followed by
phenol extractions (lane 2), and by the new filter-sup-
ported method (lane 3). Two probes each were prepared.
In case of SDS lysis/phenol extraction (lane 2), RNase
was added to the EcoRI restriction digest to remove
RNA. The isolates were applied onto a 1% agarose gel
and electrophorized in TAE buffer. Figures indicate posi-
tions of molecular weight markers (X Hind111 and Phi
X 174 HueIII).
with that of conventional methods, X phage
DNA was isolated from liquid cultures both
by a two-step cesium chloride gradient puri-
fication followed by formamide extraction of
the phage DNA (1,3) and by a SDS-lysis
method of phages with subsequent phenol ex-
tractions of the released DNA (2). The DNA
prepared by all three methods is similar; i.e.,
upon agarose gel electrophoresis the XgtlO
arms at 32,7 10 and at 10,630 bp as well as
the 604-bp MCI insert are discernible in each
case (Fig. 3). The recovery was comparable
with all three methods, i.e., more than 80%,
while the purity differed somewhat. Purity
was best with the cesium cholide gradient
method (lane 1). DNA isolated by the
200 JAKOBI, WIEMANN.
SDS-lysis method (lane 2) contained large
amounts of RNA which had to be removed
by adding RNase to restriction digests. The
DNA isolated by the new filter-supported
method was not totally pure (lane 3). How-
ever, it was pure enough to be as successfully
used for preparative purposes as the DNA ob-
tained by the other two methods.
Preparative Usage of Isolated DNA:
Recloning of Insert DNA
For subcloning, Xgt 1OMCI DNA obtained
by the filter-supported method was digested
with EcoRI, the restriction fragments were
separated by electrophoresis in low melting
agarose, and the 604-bp MCI band was cut
out. The gel slice was melted at 68°C and
cooled to 37°C MCI DNA religated into de-
phosphorylated Xgt 10 DNA, and the phages
plated out on E. coli C600. To ensure that
inserted DNA was really MCI DNA and not
any contaminating bacterial DNA of compa-
rable size, a nylon replica from the plate was
hybridized to a biotinylated plasmid contain-
ing the MCI insert. Only the clear plaque-
forming units exhibiting positive signals con-
tained an insert as proved by concomitant
DNA isolation and restriction analysis (data
not shown). Also, transformation of plasmid
vectors with a subcloned insert (ligated as
above) was found possible. This makes un-
ambiguously clear that the filter-supported
DNA preparation is a reliable method for
preparative purposes.
DISCUSSION
The separation of bacterial DNA from the
phage DNA can conveniently be performed
at a stage where the phages are intact. At this
stage, bacterial DNA is digestable selectively
with DNase. The DNase, however, has to be
removed afterward by phenol extraction (2).
To avoid working with this poisonous and
caustic material (11) and to shorten working
time, we attempted to remove the bacterial
nucleic acids chromatographically by the
DEAE cellulose procedure (4). This removed
AND PYERIN
in addition contaminants originating from
the agar growth medium which considerably
inhibit many of the DNA-modifying en-
zymes hindering preparative usage of the
phage DNA. The DEAE cellulose treatment
therefore offers the advantage of working also
with plate lysates providing higher phage ti-
ters than liquid cultures.
After the bacterial nucleic acids are re-
moved, the phages are precipitated. Precipi-
tation by acetic acid as described for phage M
13 (7) or with ethanol and acetone resulted in
very low recoveries. We therefore returned to
the conventional polyethylene precipitation
procedure and found it optimal in recovery
and speed. At final concentrations of 5%
polyethylene glycol and above, recoveries of
phage DNA have been better than 80% and
none of the time-consuming incubations on
ice or at temperatures below the freezing
point are necessary. The precipitated phages
can rather immediately be immobilized on
glass fiber filters, in which state the release of
DNA is accomplished.
Formamide treatment of phage X appears
to cause the DNA in the phage to be ejected
from the tail (1). On the other hand, DNA has
been shown to bind to glass fiber filters in the
presence of high concentrations of sodium
perchlorate (7- 10). Combining these obser-
vations, the immobilized phages were treated
by 50% formamidef4 M sodium perchlorate
solution and found to dissociate phage DNA
from phage protein, the DNA being immedi-
ately bound to the filters upon release,
whereas proteins are unable to bind under
such conditions. This makes it possible to de-
proteinize and desalt the DNA conveniently
and quickly. Deproteinization is achieved by
adding 4 M sodium perchlorate as washing so-
lution to the filter-bound DNA followed by a
short low-speed centrifugation. The depro-
teinization in solution with organic solvents
such as phenols or chloroform (2) with all
their undesirable implications if inhaled are
omitted as is the rather expensive cesium
chloride gradient ultracentrifugation ( 1,3).
Desalting of the filter-bound DNA is carried
 PREPARATION
out similarly. A short wash with 70% ethanol
is sufficient. Ethanol, a known inhibitor of
enzymes for the further processing of DNA,
is removed quantitatively from the filters
simply by evaporation and checking by
smelling. Desalting and concentration by eth-
anol precipitation, a necessary step in con-
ventional procedures ( l-4), becomes super-
fluous. In fact, the possibility of carrying out
all the necessary washing steps so easily,
quickly, and effectively on filter plates is the
major advantage of this method. Since the
elution of DNA from the filters is again easily
accomplished by adding a small volume of
solution or of water followed by a short incu-
bation and low-speed centrifugation, the
method allows the rapid preparation of large
numbers of probes simultaneously. X phage
DNA from series of 5-ml liquid cultures and
90-mm plate lysates was obtained in quanti-
ties of approximately 2 (phage titer 10” pfu/
ml) and 10 pg, respectively, in less than 2 h
and with high reproducibility. Moreover, the
purity of the resulting DNA preparations
was, without exception, suitable for restric-
tion analysis and subcloning. The filter-sup-
ported preparation of phage DNA, therefore,
meets all the requirements for automation or
semiautomation.
The filter-supported preparation yields
DNA in quantity and quality comparable to
that obtained by other reliable methods (l-3)
but incomparably faster. It is as fast a method
as those employed for analytical purposes
(1,4). The method is also admirably cheap,
using plastic ware, short low-speed centrifu-
gation, and inexpensive solutions.
OF X PHAGE DNA 201
ACKNOWLEDGMENTS
We are grateful to Prof. V. Kinzel for his continuous
encouragement and support, Dr. J. Reed for semantic
assistance, and M. Kaszkin for help in computer graph-
ics. This work was supported in part by the Sonderver-
mijgen des Deutschen Krebsforschungszentrums and the
Deutsche Forschungsgemeinschat?.
REFERENCES
1. Davis, R. W., Botstein, D., and Roth, J. R. (1980)
Advanced Bacterial Genetics: A Manual for Ge-
netic Engineering, Cold Spring Harbor Labora-
tory, Cold Spring Harbor, NY.
2. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982)
Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory. Cold Spring Harbor,
NY.
3. Glover, D. M. ( 1985) DNA Cloning, Vol. 1, IRL
Press, Chicago.
4. Silhavy, T. J., Berman, M. L., and Enquist, L. W.
(1984) Experiments with Gene Fusion, Cold
Spring Harbor Laboratory, Cold Spring Harbor,
NY.
5. Uhler, M. D., Carmichael, D. F., Lee, D. C., Chrivia,
J. C., Krebs, E. G., and M&night, G. S. (1986)
Proc. Natl. Acad. Sci. USA 83, 1300- 1304.
6. HamesB. D., and Higgins, S. J. (Eds.) (1985) Nucleic
Acid Hybridization: A Practical Approach, IRL
Press, Chicago.
Kristensen, T., Voss, H., and Ansorge, W. (1987)
NucleicAcids Res. 15,5507-t&16.
Chen, C. W., and Thomas, C. A., Jr. (1980) Anal.
Biochem. 101,339-341.
Yang, R. C.-A., Lis, J., and Wa, R. (1979) in Meth-
ods in Enzymology (Wu, R., Ed.), Vol. 68, pp.
176- 182, Academic Press, New York.
10. Marko, M. A., Chipperlield, R., and Bimbaum,
H. C. (1982) Anal. Biochem. 121,382-387.
11. Stecher, P. G., Windholz, M., and Leahy, D. S.
(Eds.) (1968) The Merck Index, Merck & Co.,
Rahway, NJ.