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A versatile quick-prep of genomic DNA from Gram-positive bacteria
Andreas Pospiech a [email protected] and Björn Neumann
[a]Andreas Pospiech and [b]Björn Neumann, Ciba-Geigy AG, Department of
Biotechnology, K-681.308, CH-4002 Basel, Switzerland, and Björn Neumann, Research
Institute of Molecular Pathology, A -1030 Vienna, Austria.
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This Technical Tip was first published in Trends in Genetics (1995) 11, 217-218
Many Gram-positive bacteria are used in industrial processes (e.g. Bacillus subtilis,
lactococci or streptomyces), and the genetic manipulation of these organisms requires the
preparation and analysis of chromosomal DNA. However, methods generally used for
isolation of chromosomal DNA from E. coli are seldom successful with Gram-positive
species, because of differences in cell-wall composition and structure between Gramnegative and -positive bacteria. There are several publications describing short protocols
for chromosomal DNA isolation from Gram-positive bacteria. However, they are often
specific to one group or even one species of microorganisms (Ref. 1, 2, 3, 4, 5, 6).
Furthermore, the isolation schemes are time-consuming, costly and often require
hazardous reagents.
The protocol we present here works for the isolation of genomic DNA from both Gramnegative bacteria (Ref. 7) and, with very small adaptions, for a broad range of Grampositive prokaryotes (Table 1). The purification scheme is simple, reproducible, fast and
safe, since hazardous solutions are omitted. The method utilizes lysozyme to break open
the cell wall. The procedure can be used for small- and large-scale preparations of
chromosomal DNA and the purified DNA is of excellent cloning quality. There is no
detectable residual nuclease activity. DNA samples obtained were checked by incubating
2 μg of DNA for 3 h at 37°C in restriction buffer. After electrophoresis there was no
obvious degradation visible compared to untreated DNA. Self-ligation of digested DNA
is quantitative ( Figure 1). Restricted and self-ligated samples showed a quantitative shift
towards higher molecular weights. The cloning efficiency was also checked in a shotgun
cloning experiment using Streptomyces lividans DNA. 1 μg of DNA gave rise to 7 ×
104 recombinant clones, analyzed by plasmid minipreparations.
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FIGURE 1. Gel electrophoresis of (a) restricted and (b) subsequently religated
chromosomal DNA from diverse Gram-positive bacteria: 1, B. subtilis; 2, B.
thuringiensis; 3, L. lactis; 4, S. lutea; 5, M. luteus; 6, A. mediteranei; 7, S. lividans; 8, S.
pilosus; 9, S. carnosus. Lanes M contain size markers: λ DNA digested with
HindIII. (a) DNA (5 mg) in lanes 1−3 and 9 was digested with EcoRI, and with
SalI in lanes 4−8. (b) Lanes contain digested DNA (2.5 mg) that was
phenol−chloroform extracted and then ligated for 4 h at 11°C.
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Protocol
1.Grow cells (Table 1) in 30 ml of rich medium like M17 (Merck), supplemented with
0.5% glucose, brain heart infusion/BHI-BBL (Becton Dickinson), 148G (Ref. 8) or LB at
30°C overnight or at 28°C for 2 d (actinomycetes).
2.Harvest cells by centrifugation (10 min, 3000 g) and resuspend in 5 ml of SET (75 mM
NaCl, 25 mM EDTA, 20 mM Tris, pH 7.5).
3.Add lysozyme to a concentration of 1 mg/ml and incubate at 37°C for 0.5−1 h.
Then add 1/10 volumes of 10% SDS and 0.5 mg/ml proteinase K and incubate at 55°C
with occasional inversion for 2 h.
4.Add 1/3 volumes 5 M NaCl and 1 volume of chloroform and incubate at room
temperature for 0.5 h with frequent inversion.
5.Centrifuge at 4500 g for 15 min and transfer the aqueous phase to a new tube using a
blunt-ended pipette tip.
6.Precipitate the DNA by adding 1 volume of isopropanol and gently invert the tube.
Transfer DNA into a microfuge tube, rinse with 70% ethanol, dry with vacuum and
dissolve in a suitable volume of TE. The amount of DNA obtained is between 1 and 5
mg.
Lysis of the cells is critical for the success of this procedure. The incubation time with
lysozyme can be extended (0.5 h represents the minimum time). However, in quite a few
streptomycete species, DNA showed evidence of degradation during longer lysozyme
treatment, presumably through the production or activation of nucleases. For these
species the incubation time should be as short as possible (T. Kieser, pers. commun.).
References
[1] dos Santos A.L.L. and Chopin A. (1987) FEMS Microbiol. Lett. , 42:209-212.
[2] Caparon M.G. and Scott J.R. (1991) Methods Enzymol. , 204:556-586. Cited by
[3] Hopwood D.A. et al. (1985) Genetic Manipulation of Streptomyces. A Laboratory
Manual, John Innes Foundation :69-80.
[4] Riele H., Michel B. and Ehrlich S.D. (1986) Proc. Natl Acad. Sci. USA, 83:25412545.
[5] Novick R.P. (1991) Methods Enzymol. , 204:587-636. Cited by
[6] Mak Y.M. and Ho K.K. (1992) Nucleic Acids Res., 20:4101-4102. Citedby
[7] Neumann B., Pospiech A. and Schairer H.U. (1992) Trends Genet., 8:332-333.
[8] Schupp T. and Divers M. (1987) FEMS Microbiol. Lett., 36:159-162.
© 1996 Elsevier Science Limited. All rights reserved.