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DOI 10.1007/s10529-005-4688-z
Received 8 June 2005; Revisions requested 16 June 2005 and August 2005; Revisions received 22 August 2005 and 19 October 2005;
Accepted 28 October 2005
Abstract
A very simple and rapid method for extracting genomic DNA from Gram-negative bacteria, Gram-positive
bacteria and yeasts is presented. In this method, bacteria or yeasts are lysed directly by phenol and the
supernatant is extracted with chloroform to remove traces of phenol. The supernatant contains DNA that is
suitable for molecular analyses, such as PCR, restriction enzyme digestion and genomic library construction.
This method is reproducible and simple for the routine DNA extraction from bacteria and yeasts.
extract, 50 g dextrose, 10 g CaCO3 in 1 l distilled sequent experiments or stored at )20 C. The
water, pH 7.0) at 30 C and 250 rpm. Pichia ano- purity and yield of the DNA were assessed spec-
mala sp. and Saccharomyces cerevisiae ATCC trophotometrically by calculating the A260/A280
18824 were cultivated in YEPD broth (5 g yeast ratios and the A260 values to determine protein
extract, 10 g peptone and 10 g dextrose in 1 l dis- impurities and DNA concentrations.
tilled water, pH 7.0) at 28 C and 250 rpm.
1–2 lg gDNA from E. coli TG1, E. coli HB101, We also tested the availability of commercial
K. pneumoniae, B. subtilis, A. tumefaciens, T. DNA extraction kit (DingGuo Biotech. Co.,
tengcongenis, G. oxydans, A. suboxydans, P. ano- Ltd., Beijing) to prepare gDNA from E. coli
mala, C. glutamicum was incubated with 5 U TG1. The mean DNA yield was 2413 lg/g dry
EcoRI in a final volume of 20 ll for 5 h at 37 C cells, with A260/A280 ratio of 1.68. Table 1
and applied to 1% agarose gel electrophoresis. showed that the recommended method was in
particular useful to extract gDNA from Gram-
Analysis of construction of genomic library negative bacteria. We also tested four other
Gram-negative bacteria including Xanthomonas
20 lg gDNA from A. suboxydans sp. was par- campestris which produced extracellular material,
tially digested by 0.5 U Sau3AI, for 4 min at xanthan gum (Becker et al. 1998), Acetobacter
30 C, in a final 80 ll volume. Then the Sau3AI- aceti, Acetobacter pastoris, and E. coli C600
digested mixture was placed into an 80 C water- using this method. The yields obtained were
bath for 20 min for heat inactivation of Sau3AI. above 3000 lg DNA/g dry cells, A260/A280 ratios
5 ll of the digestion mixture was then ligated with were between 1.57 to 1.74 (data not shown).
100 ng of BamHI-digested and dephosphorylated However, the gDNA yields from Gram-positive
vector pBluescript II SK(-) in a 20 ll volume at bacteria and yeasts were not so high due to the
16 C for 18 h. The ligation mixture was trans- incomplete lysis of cell wall using phenol as the
formed into competent E. coli strain TG1. The sole lysis reagent. But the gDNA yields obtained
transformation conditions was: 30 min on ice, from two yeasts were higher than those obtained
42 C for 2 min, 2 min on ice, 300 ll SOC broth from two Gram-positive bacteria; the reason
(20 g tryptone, 5 g yeast extract, 0.5 g NaCl, might be the use of acid-washed glass beads
0.2 g KCl, 2 g MgCl6ÆH2O, 5 g MgSO4Æ7H2O, 4 g while extracting gDNA from yeasts. However,
dextrose in 1 l distilled water, pH 7.0) was added this method was still suitable for preparation of
and incubated at 37 C for 1 h. The cells were gDNA from Gram-positive bacteria and yeasts
plated on solid minimal medium (5 g Na2H- due to its extreme rapidness and cost-effective-
PO4Æ12H2O, 0.9 g KH2PO4, 0.16 g NaCl, 2 g ness, and the DNA yield was high enough for
NH4Cl, 0.5 g MgSO4Æ7H2O, 0.01 g CaCl2, 4 g
D-arabitol, 15 g agar in 1 l distilled water, pH 7.0)
with 100 lg ampicillin/ml and incubated at 37 C
Table 1. Yields and quality of DNA obtained by using the
for 72 h. Transformants were further tested for recommended method.
growth in liquid minimal medium with 50 lg
ampicillin/ml at 37 C and 250 rpm for 72 h. Microorganism Mean DNA Yielda A260/A280
(lg/g dry cells)
2–4 h (except for DNA extraction using DNA Innovation Project from the Chinese Academy of
extraction kit which could complete the process Sciences.
within 1 h in the case of several samples). Sec-
ond, the method yielded equally high molecular
weight DNA compared with standard phenol/
chloroform protocol. Third, this method was References
cost-effective, since it only used phenol, chloro-
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In conclusion, we have presented here a DNA
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extraction method that is easy to use, rapid, cost- Niemi RM, Heiskanen I, Wallenius K, Lindstrom K (2001)
effective and applicable for many microorgan- Extraction and purification of DNA in rhizosphere soil
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Acknowledgements Syn CK, Swarup S (2000) A scalable protocol for the isolation
of large-sized genomic DNA within an hour from several
We are very grateful to Dr. Bernhard Seiboth bacteria. Anal. Biochem. 278: 86–90.
(Institute of Chemical Engineering, Vienna Uni- Wilson K (1990) Preparation of genomic DNA from bacteria.
In: Ausubel FM & Brent R eds. Current Protocols in
versity of Technology, Austria) for valuable dis- Molecular Biology, New York: Greene Publ. Assoc. and
cussion and critically reading the manuscript. We Wiley Interscience, pp. 241–245.
also thank Ms. Yu Bai for the preparation of the Xue Y, Xu Y, Liu Y, Ma Y, Zhou P (2001) Thermoanaerobacter
tengcongensis sp. nov., a novel anaerobic, saccharolytic,
manuscript. The research was supported by the
thermophilic bacterium isolated from a hot spring in
National Basic Research Program of China (No. Tengcong, China. Int. J. Syst. Evol. Microbiol. 51: 1335–
2004 CB 719002) and the National Knowledge 1341.