alized on polyvinylidene difluoride membranes by transillumination. Appl. Theor.
Electrophor. 1:59-60.
5.Smejkal, G.B. and H.F. Hoff. 1993. Co-localization of molecular mass marker proteins on
Western blots. BioTechniques 15:796-798.
6.Smejkal, G.B. and H.F. Hoff. 1994. Filipin
staining of lipoproteins in polyacrylamide
gels: sensitivity and photobleaching of the fluorophore and its use in a double staining
method. Electrophoresis 15:922-925.
7.Van Dam, A.P. 1994. Transfer and blocking
conditions in immunoblotting, p. 79-80. In
B.S. Dunbar (Ed.), Protein Blotting: A Practical Approach. IRL Press, Oxford.
The authors gratefully acknowledge
John Shainoff, Patricia DiBello, Ruth Earley and June O’Neil for internal review of
this manuscript. This work was funded by
National Institutes of Health Grant HL43339. Address correspondence to Henry F.
Hoff, Cleveland Clinic Foundation Research Institute, Department of Cell Biology NC-1, 9500 Euclid Avenue, Cleveland,
OH 44195, USA.
Received 29 November 1995; accepted
26 January 1996.
Gary B. Smejkal, Rudolf M.
Snajdar and Henry F. Hoff
The Cleveland Clinic
Foundation Research Institute
Cleveland, OH, USA
Alternative Method for
Isolation of DoubleStranded Template for
DNA Sequencing
BioTechniques 21:233-235 (August 1996)
The increased use of plasmid DNAs
in recombinant DNA technology, coupled with the growing popularity of
double-stranded DNA sequencing, necessitates the development of rapid, inexpensive and efficient procedures for
the isolation of highly purified plasmids. Numerous miniprep procedures
have been described for preparing plasmid DNAs (1,2,4,5,8,9). A quick and
efficient alternative method for the
Vol. 21, No. 2 (1996)
preparation of large quantities of purified plasmid DNA is presented in this
report. Following alkaline lysis of the
bacterial cells, polyethylene glycol6000 (PEG-6000) (13% final concentration) is used to precipitate the nucleic acid. The plasmid is then further
enriched by selective solubilization using 2.75 M LiCl. Any remaining chromosomal DNA, RNA and cellular debris are removed at this step. This
method does not involve any phenolchloroform extractions or treatment of
the samples with ribonuclease and requires only two hours for completion of
the protocol following growth of the
bacterial cells. The quality and yield of
the plasmid obtained with this method
is comparable to that isolated by cesium chloride-ethidium bromide gradient
centrifugation (CsCl-EtdBr) or purified
through QIAGEN QIAprep® columns
(Qiagen, Chatsworth, CA, USA). The
plasmid obtained is amenable to digestion with various restriction endonucleases, can be used for cloning with high
efficiency and is also suitable as a template for dideoxy sequencing. This inexpensive procedure, which includes a
PEG-precipitation step, represents a
modification of the alkaline lysis
method (1,5) and the LiCl solubilization method (3) (Table 1).
For denaturation of the DNA template prior to dideoxy sequencing, a
volume of 18 µL of plasmid DNA, as
isolated in Table 1, was mixed with 2
µL 2 M NaOH. After 5 min, the solution was neutralized by the addition of
8 µL 5.5 M LiCl. The DNA was precipitated with 75 µL of cold 100% ethanol
at -70°C for 10 min and recovered by
centrifugation at 4°C for 10 min at
14 000× g. The DNA pellet was rinsed
with 100 µL of cold 70% ethanol, dried
under a vacuum for 5 min and stored as
a dry pellet at -20°C (stable for several
weeks). For the sequencing reaction,
the denatured plasmid template was resuspended in a mixture of annealing
buffer and sequencing primer (primer
T3) as described in the standard Sequenase® protocol provided by United
States Biochemical (Cleveland, OH,
USA) (7).
For comparison purposes, pBluescript® II SK(+) plasmid (Stratagene,
La Jolla, CA, USA) was isolated by the
method described above and two other
Benchmarks
Table 1. The Procedure for the Rapid Isolation of Plasmid DNA
1. Inoculate a single bacterial colony into 1.5 mL of TB medium [17 mM
KH2PO4, 72 mM K2HPO4, 1.2% (wt/vol) Bacto-Tryptone, 2.4% (wt/vol) bacto-yeast extract and 0.4% glycerol] in a 10–15-mL culture tube and incubate
in the presence of the appropriate antibiotic. Incubate at 37°C in a shaker-incubator for 12–18 h.
2. Centrifuge the bacterial cells in a microcentrifuge tube at 14 000× g for 2 min.
3. Remove the supernatant by aspiration, resuspend the bacterial pellet in 100
µL of GTE buffer (50 mM glucose, 25 mM Tris-HCl, pH 8.0, and 10 mM
EDTA) and incubate at room temperature for 5 min.
4. Chill the pellet on ice, add 200 µL of freshly prepared alkaline lysis solution
(0.2 N NaOH, 1% sodium dodecyl sulfate [SDS]), mix by inversion and incubate on ice for 5 min. For bacterial cells, which are difficult to lyse, a 5-min
pretreatment with lysozyme at room temperature is optional (1 mg/mL final
concentration).
5. Neutralize the solution by adding 150 µL of 3 M sodium acetate, pH 4.8, mixing by inversion and incubating on ice for 5 min.
6. Centrifuge the mixture at 14 000× g for 10 min at 4°C and transfer the supernatant to a clean microcentrifuge tube.
7. Add 145 µL of 40% PEG-6000 to the supernatant and mix by inversion. Incubate on ice for 10 min.
8. Centrifuge the plasmid DNA at 4°C for 10 min at 14 000× g.
9. Remove the supernatant by aspiration and perform a second brief spin to collect and remove all the supernatant.
10. Dissolve the centrifuged plasmid DNA in 100 µL of deionized H2O or TE
buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5), add 100 µL 5.5 M LiCl and incubate on ice for 10 min.
11. Centrifuge the sample at 14 000× g for 10 min at 4°C. Discard the pellet and
transfer the supernatant to a clean tube.
12. Precipitate the plasmid DNA by adding 0.6 vol of isopropanol. Incubate for 10
min at room temperature.
13. Centrifuge the DNA at 14 000× g for 10 min at 4°C. Remove supernatant.
14. Rinse the DNA pellet with 500 µL of cold 70% ethanol. Dry the DNA pellet under a vacuum for 5 min.
15. Dissolve the DNA pellet in 20 µL of TE buffer. Load 1 µL of total volume on a
1% agarose gel to analyze the quantity and quality of plasmid DNA.
methods, including the standard alkaline lysis procedure, followed by cesium chloride gradient centrifugation
(4) and QIAGEN QIAprep purification
using manufacturers’ recommended
protocols.
In this report, we have described a
simple procedure to prepare high-quality plasmid DNA that can be used for
most molecular biological techniques.
We routinely obtained 3–5 µg plasmid
DNA per 1.5 mL bacterial culture. This
relatively high yield of plasmid DNA is
aided by the use of TB medium, which
allows high-density bacterial growth as
described by Tartof and Hobbs (6). For
low-copy number plasmids, such as
pBR322, a 5-mL culture may be necessary for the isolation of sufficient plasmid for further analysis.
234 BioTechniques
Two important parameters in this
procedure for obtaining supercoiled
plasmid DNA (>95%) are to keep the
samples on ice and to avoid vortex mixing. Additionally, this procedure efficiently removes bacterial RNA by a
combination of alkaline treatment and
selective precipitation with LiCl. This
approach avoids the need for ribonuclease treatment and subsequent organic
extractions.
Figure 1 compares the quality and
conformation of plasmid pBluescript II
SK+ DNA isolated by our modified
PEG-precipitation method and plasmid
isolated by CsCl–EtdBr-gradient centrifugation and the commercial QIAGEN QIAprep column. The plasmid
DNA isolated by our procedure is primarily supercoiled and shows no chro-
mosomal DNA contamination; the
quality and yield are as good as or better than that produced by the two other
methods compared in this analysis. The
Figure 1. Comparison of plasmid DNAs
[pBluescript II SK(+)] isolated using three different methods. Lane 1: λ bacteriophage DNA
digested with EcoRI and HindIII. Lane 2: plasmid purified using CsCl-EtdBr gradient centrifugation. Lane 3: plasmid isolated using QIAGEN
QIAprep. Lanes 4–8: individual plasmid isolations using our modified PEG-precipitation
protocol.
Figure 2. Autoradiograph of DNA sequencing
reactions separated on an 8% denaturing
polyacrylamide gel. Dideoxy termination reactions are indicated by G, A, T or C. Doublestranded pBluescript II SK+ DNA templates were
isolated by the following methods: Lane 1: CsClEtdBr gradient centrifugation. Lane 2: QIAGEN
QIAprep. Lane 3: modified PEG-precipitation
protocol.
Vol. 21, No. 2 (1996)
plasmid isolated by our procedure can
be used directly as a template for double-stranded DNA sequence analysis.
Figure 2 shows nucleotide sequence
data from the multiple cloning site region of plasmid pBluescript II SK(+).
Sequencing reactions using template
isolated by our procedure generated sequencing information of equal quality
to those templates isolated by the CsCl
or the QIAGEN procedures.
In conclusion, we report here a rapid
and inexpensive protocol for plasmid
isolation. Plasmid isolated by this
method is of sufficient quality for use
in DNA sequencing reactions and other
molecular biological techniques. This
procedure provides an attractive alternative to more expensive and/or timeconsuming methods currently used to
prepare plasmid DNA.
REFERENCES
1.Birnboim, H.C. and J. Doly. 1979. A rapid
alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res.
7:1513-1523.
2.Del Sal, G., G. Manfioletti and C. Schneider. 1988. A one tube plasmid DNA minipreparation suitable for sequencing. Nucleic
Acids Res. 16:9878.
3.He, M., A. Wilde and M.A. Kaderbhai.
1990. A simple single-step procedure for
small-scale preparation of Escherichia coli
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4.Maniatis, T., E.F. Fritsch and J. Sambrook.
1982. Large scale isolation of plasmid DNA,
p. 86-95. Molecular Cloning: A Laboratory
Manual. Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY.
5.Stephen, D., C. Jones and J.P. Schofield.
1990. A rapid method for isolating high quality plasmid DNA suitable for DNA sequencing. Nucleic Acids Res. 18:7463.
6.Tartof, K.D. and C.A. Hobbs. 1988. New
cloning vectors and techniques for easy and
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7.United States Biochemical Corporation.
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DNA sequencing. 5th ed. p. 6-9, Cleveland,
OH.
8.Wang, L.M., D.K. Weber, T. Johnson and
A.Y. Sakaguchi. 1988. Supercoil sequencing
using unpurified templates produced by rapid
boiling. BioTechniques 6:839-843.
9.Ziai, M.R., C.V. Hamby, R. Reddy, K.
Hayashibe and S. Ferrone. 1989. Rapid purification of plasmid DNA following acid precipitation of bacterial proteins. BioTechniques
7:147.
The authors would like to thank Kecia
D. Carlson, Doug Smart and Gabriele Linden for technical and editorial support of
this work. This work is supported by the
Vol. 21, No. 2 (1996)
National Science Foundation under EPSCoR Grant No. 4752-22. This work also received matching support from the State of
Kansas EPSCoR Program. Address correspondence to Alan Taylor, Department of
Biological Sciences, Molecular Biology
Core Laboratory, Wichita State University,
1845 Fairmount, Box 26, Wichita, KS
67260-0026, USA. Internet: mtaylor@twsu.
vm.uc.twsu.edu
Received 6 November 1995; accepted
26 January 1996.
Sagypash Sadiev and Alan
Taylor
Wichita State University
Wichita, KS, USA