Nucleic Acids Research, Vol. 20, No. 15 4101-4102
An improved method for the isolation of chromosomal DNA from various bacteria and cyanobacteria Y.M.Mak and K.K.Ho Department of Botany, Faculty of Science, National University of Singapore, Lower Kent Ridge Road, 0511 Singapore Submitted April 22, 1991 Although many methods of isolating chromosomal DNA exist, all are inefficient to some degree (1). Cell lysis is often timeconsuming, and can result in damaged DNA. This paper describes the use of two solvents, n-butanol and phenol, in the isolation of DNA from a variety of bacteria and cyanobacteda (Listed in Table 1). The method is rapid, high-yielding, and has been used previously for the detection of recombinant plasmids in Escherichia coli (2). It has now been extended to the isolation of chromosomal DNA and found to be particularly useful for small scale preparation. All bacterial strains were grown overnight in LB media with the exception of Agrobacterium which, because of its slow growth, was left to grow for two days. Axenic cultures of the cyanobacteria, Spirulina and Oscillatoria, grown in SOT (3) and Bold's media (4) respectively, were used. 500 da aliquots of the bacterial cultures were subjected to the butanol extraction without further treatment. However, in the case of the cyanobacteria, Table 1. Yield and quality of total nucleic acids (NA) obtained from 500 or phenol extractions with or without SDS supplement. Specimens
1.
2.
A,,/
NA extracted with phend
'Visualized
Total NA
Am'
on EtBrgel
(ig)
on EtBrgel
NA extracted with 0.1% SDS and phenol Am' Visualized A on EtBrgel (Wg)
1.7
4J
18
1.5
4
1.5
VI
70
1.8
V
4
14
1.9
4
21
1.9
4
25
1.6
4
1.5
x
NA extracted with 0.1% SDS and butanol Total NA Visualized A,.J
A,,
Visualized on EtBrgel
(W)
Total NA
(WAg)
A
Escherichia coli (JM 109)
65
1.5
VI
45
2.0
V
20
Pseudomonas aeruginosa
67
1.4
V
60
1.9
4
43
Rhizobium sp.
43
1.5
4
26
1.8
4
71
1.4
Agrobacterium tumefaciens
29
2.0
x
22
1.8
4
31
2.0
x
Agrobacterium rhizogenes
22
2.1
x
24
1.9
/
28
1.8
x
41
1.7
x
22
1.7
x
27
2.0
x
28
Am,
Bacteria strains (Gram -ve)
Bacteria strains (Gram +ve)
Staphylococcus aureus
3.
Id (4 mg wW) of bacterial cultures and 30 mg wW of cyanobacterial cultures, using butanol
NA extracted with butand Total NA
chains of Oscillatoria were fragmented, then they, along with Spirulina, were mixed with one-third volume of ethanol, pelleted and resuspended in 500 Al LB medium. Aliquots of the cultured cells were mixed by vortexing with 1.5 volumes of n-butanol and centrifuged. DNA from the lower aqueous phase was removed and precipitated with ethanol. It was then recovered by centrifugation, washed in 70% ethanol and vacuum dried. RNA collected along with the DNA could be eliminated with RNase treatment during restriction of DNA. N-butanol in the initial step may be replaced by 1.0 volume of water-saturated phenol (supplemented with 0. 1 % 8-hydroxyquinoline) without any loss in yield of DNA and with the advantage of removing more protein contaminants (as shown by the A260/A280 ratios) than the butanol extraction. However, phenol left in solution has been shown to inhibit restriction of phenol-extracted DNA from some bacteria strains. In the case of Pseudomonas aeruginosa, DNA extracted from both phenol
Lactobacillusacidophilius
4
1.5
V
6
1.7
4
16
1.5
4
7
1.4
4
Bacillus subtilis
14
1.2
V
15
1.8
V
4
1.3
4
6
1.7
4
Micrococcus luteus
38
1.8
4
22
1.9
4
31
1.8
4
19
1.9
V
Streptococcus faecalis
29
1.8
VI
21
1.6
4
27
1.8
V
23
2.0
4
Spirulina platensis
55
1.4
4
12
1.9
4
3
1.7
4
3
2.4
4
Oscillatona sp.
23
1.9
x
22
1.9
4
32
1.6
V
32
1.8
4
Cyanobacteria strains (Gram -ve)
*The state of the isolated DNA was checked by subjecting 80% of the materials to agarose gel electrophoresis. The symbols, \/ and x, represent undegraded and degraded forms, respectively.
4102 Nucleic Acids Research, Vol. 20, No. 15
ACKNOWLEDGEMENTS
and butanol methods is restrictable by enzymes (Figure 1). Figure 2 shows the quality of chromosomal DNA isolated from Bacillus subtilis and Streptococcus faecalis. These two bacteria strains are known to be involved in hospital acquired infection. Chromosomal DNA was successfully extracted from most of the microorganisms listed in Table 1 using the butanol and/or phenol extractions. Exceptions include Agrobacterium and Oscillatoria where chromosomal DNA was isolated using phenol but not butanol extraction. Both methods failed to isolate DNA from Staphylococcus aureus. In general, repeated butanol extractions increased the final yield of DNA. In an effort to improve on the method, a modified procedure was developed which involved the addition of 5 [LIO 1% Sodium Dodecyl Sulphate (SDS) to 500 A1 aliquots of cultured cells prior to the butanol/phenol extractions. This enabled DNA to be isolated from all the specimens except Staphylococcus aureus which required treatment with 0.5% or more SDS. DNA from Agrobacterium was isolated with 0.5 % SDS supplement using butanol extraction. LB medium, supplemented with 0.1 % SDS, proved an efficacious extraction buffer, inhibiting DNase activity. Indeed, it yielded the most DNA when compared to such commonly used buffers as TE (10 mM Tris-HCI, pH 8.0, 1 mM EDTA), STE (TE with 100 mM NACI), water and miniprep lysis buffer (25 mM Tris-HCI, pH 8.0, 10 mM EDTA, 50 mM glucose). The yields of DNA listed in Table 1 have been calculated from the absorbance of the solutions at 260 nm. The yield of DNA was also estimated by a comparison of the isolated chromosomal DNA with a molecular weight DNA standard when both were run on a gel stained with ethidium bromide. This latter estimation has shown that the absorbance-based values were often gross overestimations which have been attributed to the presence of large amounts of RNA and protein contaminants. The butanolextracted DNA, therefore, would have produced a larger overestimation, since it contained more protein contaminants. The gel-visualisation method supported the efficacy of the modified protocol (including SDS treatment); the higher yield of chromosomal DNA was not shown, however, by the absorbance-based method. The DNA extraction procedure mentioned above may be scaled up safely. Large volumes of saturated cell cultures may be concentrated by centrifugation and resuspended in as small a volume as one-hundredth of the original volume of LB (Table 2) and then extracted by any one of the methods listed in Table 1. The DNA obtained by such large preparation was suituable
We acknowledge the help of Dr K.Gamini in acquiring some of the bacteria strains studied here. The research is supported by a NUS grant.
REFERENCES 1. Bollet,C., Gevaudan,M.J., de Lamballerie,X., Zandotti,C. and de Micco,P. (1991) Nucleic Acids Res. 19, 1955. 2. Mak,Y.M., Sornarajah,R. and Ho,K.K. (1991) BioTechniques 11, 723. 3. Ogawa,T. and Terui,G. (1 970) J. Ferment. Technol. 48, 361 -367. 4. Nichols,H.W. and Bold,H.C. (1965) J. Phvcol. 1, 34-38.
Figure 1. Electrophoresis of Pseudomonas aeruginosa DNA restricted by various enzymes. Lane 1: 0.4 jg 1 kb DNA ladder (BRL); Lanes 2, 3 and 4: DNA extracted with butanol and restricted with PstI (P), EcoRI (E) and KpnI (K), respectively; Lanes 5, 6 and 7: DNA extracted with 0.1 % SDS and butanol, and restricted with P, E and K, respectively; Lanes 8, 9 and 10: DNA extracted with phenol and restricted with P, E and K, respectively; Lanes 11, 12 and 13: DNA extracted with 0. 1% SDS and phenol, and restricted with P, E and K respectively; Lanes 14, 15 and 16: DNA extracted with 0.5% SDS and phenol, and restricted with P, E and K, respectively; Lane 17: unrestricted DNA; and Lane 18: 0.3 /sg high molecular weight DNA marker (BRL).
Figure 2. Electrophoresis of Bacillus and Streptococcus DNA isolated by butanol and phenol extractions. 80% of total nucleic acids isolated from 500 pl of cells were electrophoresed on 0.5% TAE agarose gel. Lanes 1, 2 and 3: Butanol extraction with 0%, 0.1% and 0.5 % SDS, respectively; Lanes 4, 5 and 6: phenol extraction with 0 %, 0.1% and 0.5% SDS, respectively. Arrow indicates plasmid band. HMW: high molecular weight DNA marker (BRL) represented by 48 502 bp, 19 399 bp and 12 220 bp.
for restriction.
Of the four bacterial strains tested, only E.coli and Bacillus subtilis provided an acceptable yield of chromosomal DNA when subjected to large scale butanol extraction.
Table 2. Yield and quality of large scale preparations of total NA. 15 ml (120 mg wW) of bacterial cells were pelleted and suspended in 150 obtained using butanol or phenol extractions with or without SDS supplement. NA extracted with butanol
Specimens
Total NA
(4g) Escherichia coli (JM 109)
140
A2W/
NA extracted with phenol
AW/
Visualized
on
Visualized EtBr gel
(gg)
A,,,
on
1.9
V
365
2.0
/
A290
Total NA
EtBr gel
NA extracted with 0.1% SDS and butanol
Al of LB.
NA was
NA extracted with 0.1%
SDS and phenol
Total NA
A2J
A20
Visualized EtBr gel
Total NA
(pg)
on
(gg)
177
1.9
V
Aa/
Visualized EtBr gel
A2M
on
336
2.0
V
Bacillus subtilis
21
1.6
V
115
2.0
V
26
16
V
44
2.0
V
Streptococcus faecalis
7
18
/
7
21
VI
15
1.7
v
15
18
V
Micrococcus luteus
6
1.9
VI
6
1.9
V
8
2.1
v
8
2.0
V
An improved method for the isolation of chromosomal DNA from various bacteria and cyanobacteria Y.M.Mak and K.K.Ho Department of Botany, Faculty of Science, National University of Singapore, Lower Kent Ridge Road, 0511 Singapore Submitted April 22, 1991 Although many methods of isolating chromosomal DNA exist, all are inefficient to some degree (1). Cell lysis is often timeconsuming, and can result in damaged DNA. This paper describes the use of two solvents, n-butanol and phenol, in the isolation of DNA from a variety of bacteria and cyanobacteda (Listed in Table 1). The method is rapid, high-yielding, and has been used previously for the detection of recombinant plasmids in Escherichia coli (2). It has now been extended to the isolation of chromosomal DNA and found to be particularly useful for small scale preparation. All bacterial strains were grown overnight in LB media with the exception of Agrobacterium which, because of its slow growth, was left to grow for two days. Axenic cultures of the cyanobacteria, Spirulina and Oscillatoria, grown in SOT (3) and Bold's media (4) respectively, were used. 500 da aliquots of the bacterial cultures were subjected to the butanol extraction without further treatment. However, in the case of the cyanobacteria, Table 1. Yield and quality of total nucleic acids (NA) obtained from 500 or phenol extractions with or without SDS supplement. Specimens
1.
2.
A,,/
NA extracted with phend
'Visualized
Total NA
Am'
on EtBrgel
(ig)
on EtBrgel
NA extracted with 0.1% SDS and phenol Am' Visualized A on EtBrgel (Wg)
1.7
4J
18
1.5
4
1.5
VI
70
1.8
V
4
14
1.9
4
21
1.9
4
25
1.6
4
1.5
x
NA extracted with 0.1% SDS and butanol Total NA Visualized A,.J
A,,
Visualized on EtBrgel
(W)
Total NA
(WAg)
A
Escherichia coli (JM 109)
65
1.5
VI
45
2.0
V
20
Pseudomonas aeruginosa
67
1.4
V
60
1.9
4
43
Rhizobium sp.
43
1.5
4
26
1.8
4
71
1.4
Agrobacterium tumefaciens
29
2.0
x
22
1.8
4
31
2.0
x
Agrobacterium rhizogenes
22
2.1
x
24
1.9
/
28
1.8
x
41
1.7
x
22
1.7
x
27
2.0
x
28
Am,
Bacteria strains (Gram -ve)
Bacteria strains (Gram +ve)
Staphylococcus aureus
3.
Id (4 mg wW) of bacterial cultures and 30 mg wW of cyanobacterial cultures, using butanol
NA extracted with butand Total NA
chains of Oscillatoria were fragmented, then they, along with Spirulina, were mixed with one-third volume of ethanol, pelleted and resuspended in 500 Al LB medium. Aliquots of the cultured cells were mixed by vortexing with 1.5 volumes of n-butanol and centrifuged. DNA from the lower aqueous phase was removed and precipitated with ethanol. It was then recovered by centrifugation, washed in 70% ethanol and vacuum dried. RNA collected along with the DNA could be eliminated with RNase treatment during restriction of DNA. N-butanol in the initial step may be replaced by 1.0 volume of water-saturated phenol (supplemented with 0. 1 % 8-hydroxyquinoline) without any loss in yield of DNA and with the advantage of removing more protein contaminants (as shown by the A260/A280 ratios) than the butanol extraction. However, phenol left in solution has been shown to inhibit restriction of phenol-extracted DNA from some bacteria strains. In the case of Pseudomonas aeruginosa, DNA extracted from both phenol
Lactobacillusacidophilius
4
1.5
V
6
1.7
4
16
1.5
4
7
1.4
4
Bacillus subtilis
14
1.2
V
15
1.8
V
4
1.3
4
6
1.7
4
Micrococcus luteus
38
1.8
4
22
1.9
4
31
1.8
4
19
1.9
V
Streptococcus faecalis
29
1.8
VI
21
1.6
4
27
1.8
V
23
2.0
4
Spirulina platensis
55
1.4
4
12
1.9
4
3
1.7
4
3
2.4
4
Oscillatona sp.
23
1.9
x
22
1.9
4
32
1.6
V
32
1.8
4
Cyanobacteria strains (Gram -ve)
*The state of the isolated DNA was checked by subjecting 80% of the materials to agarose gel electrophoresis. The symbols, \/ and x, represent undegraded and degraded forms, respectively.
4102 Nucleic Acids Research, Vol. 20, No. 15
ACKNOWLEDGEMENTS
and butanol methods is restrictable by enzymes (Figure 1). Figure 2 shows the quality of chromosomal DNA isolated from Bacillus subtilis and Streptococcus faecalis. These two bacteria strains are known to be involved in hospital acquired infection. Chromosomal DNA was successfully extracted from most of the microorganisms listed in Table 1 using the butanol and/or phenol extractions. Exceptions include Agrobacterium and Oscillatoria where chromosomal DNA was isolated using phenol but not butanol extraction. Both methods failed to isolate DNA from Staphylococcus aureus. In general, repeated butanol extractions increased the final yield of DNA. In an effort to improve on the method, a modified procedure was developed which involved the addition of 5 [LIO 1% Sodium Dodecyl Sulphate (SDS) to 500 A1 aliquots of cultured cells prior to the butanol/phenol extractions. This enabled DNA to be isolated from all the specimens except Staphylococcus aureus which required treatment with 0.5% or more SDS. DNA from Agrobacterium was isolated with 0.5 % SDS supplement using butanol extraction. LB medium, supplemented with 0.1 % SDS, proved an efficacious extraction buffer, inhibiting DNase activity. Indeed, it yielded the most DNA when compared to such commonly used buffers as TE (10 mM Tris-HCI, pH 8.0, 1 mM EDTA), STE (TE with 100 mM NACI), water and miniprep lysis buffer (25 mM Tris-HCI, pH 8.0, 10 mM EDTA, 50 mM glucose). The yields of DNA listed in Table 1 have been calculated from the absorbance of the solutions at 260 nm. The yield of DNA was also estimated by a comparison of the isolated chromosomal DNA with a molecular weight DNA standard when both were run on a gel stained with ethidium bromide. This latter estimation has shown that the absorbance-based values were often gross overestimations which have been attributed to the presence of large amounts of RNA and protein contaminants. The butanolextracted DNA, therefore, would have produced a larger overestimation, since it contained more protein contaminants. The gel-visualisation method supported the efficacy of the modified protocol (including SDS treatment); the higher yield of chromosomal DNA was not shown, however, by the absorbance-based method. The DNA extraction procedure mentioned above may be scaled up safely. Large volumes of saturated cell cultures may be concentrated by centrifugation and resuspended in as small a volume as one-hundredth of the original volume of LB (Table 2) and then extracted by any one of the methods listed in Table 1. The DNA obtained by such large preparation was suituable
We acknowledge the help of Dr K.Gamini in acquiring some of the bacteria strains studied here. The research is supported by a NUS grant.
REFERENCES 1. Bollet,C., Gevaudan,M.J., de Lamballerie,X., Zandotti,C. and de Micco,P. (1991) Nucleic Acids Res. 19, 1955. 2. Mak,Y.M., Sornarajah,R. and Ho,K.K. (1991) BioTechniques 11, 723. 3. Ogawa,T. and Terui,G. (1 970) J. Ferment. Technol. 48, 361 -367. 4. Nichols,H.W. and Bold,H.C. (1965) J. Phvcol. 1, 34-38.
Figure 1. Electrophoresis of Pseudomonas aeruginosa DNA restricted by various enzymes. Lane 1: 0.4 jg 1 kb DNA ladder (BRL); Lanes 2, 3 and 4: DNA extracted with butanol and restricted with PstI (P), EcoRI (E) and KpnI (K), respectively; Lanes 5, 6 and 7: DNA extracted with 0.1 % SDS and butanol, and restricted with P, E and K, respectively; Lanes 8, 9 and 10: DNA extracted with phenol and restricted with P, E and K, respectively; Lanes 11, 12 and 13: DNA extracted with 0. 1% SDS and phenol, and restricted with P, E and K respectively; Lanes 14, 15 and 16: DNA extracted with 0.5% SDS and phenol, and restricted with P, E and K, respectively; Lane 17: unrestricted DNA; and Lane 18: 0.3 /sg high molecular weight DNA marker (BRL).
Figure 2. Electrophoresis of Bacillus and Streptococcus DNA isolated by butanol and phenol extractions. 80% of total nucleic acids isolated from 500 pl of cells were electrophoresed on 0.5% TAE agarose gel. Lanes 1, 2 and 3: Butanol extraction with 0%, 0.1% and 0.5 % SDS, respectively; Lanes 4, 5 and 6: phenol extraction with 0 %, 0.1% and 0.5% SDS, respectively. Arrow indicates plasmid band. HMW: high molecular weight DNA marker (BRL) represented by 48 502 bp, 19 399 bp and 12 220 bp.
for restriction.
Of the four bacterial strains tested, only E.coli and Bacillus subtilis provided an acceptable yield of chromosomal DNA when subjected to large scale butanol extraction.
Table 2. Yield and quality of large scale preparations of total NA. 15 ml (120 mg wW) of bacterial cells were pelleted and suspended in 150 obtained using butanol or phenol extractions with or without SDS supplement. NA extracted with butanol
Specimens
Total NA
(4g) Escherichia coli (JM 109)
140
A2W/
NA extracted with phenol
AW/
Visualized
on
Visualized EtBr gel
(gg)
A,,,
on
1.9
V
365
2.0
/
A290
Total NA
EtBr gel
NA extracted with 0.1% SDS and butanol
Al of LB.
NA was
NA extracted with 0.1%
SDS and phenol
Total NA
A2J
A20
Visualized EtBr gel
Total NA
(pg)
on
(gg)
177
1.9
V
Aa/
Visualized EtBr gel
A2M
on
336
2.0
V
Bacillus subtilis
21
1.6
V
115
2.0
V
26
16
V
44
2.0
V
Streptococcus faecalis
7
18
/
7
21
VI
15
1.7
v
15
18
V
Micrococcus luteus
6
1.9
VI
6
1.9
V
8
2.1
v
8
2.0
V
