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Small-scale purification of bacteriophage λ DNA by an airfuge centrifugation step in cesium chloride gradients
80 1988, Gene Anal Techn 5:80-82 f-
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Small-Scale Purification of Bacteriophage h DNA by an Airfuge Centrifugation Step in Cesium Chloride Gradients
gradients, rapid isolation of biologically active, highly purified DNA was accomplished starting from b a c t e r i o p h a g e h g t l l grown on agarose plates. Small amounts of several samples of bacteriophage h DNA can be purified in a single day. The recovered DNA is devoid of RNA and DNA contaminants and is efficiently cut by restriction endonucleases.
MARC MIRANDE, M Y R I A M L A Z A R D , and
Materials and Methods
JEAN-PIERRE WALLER
Preparation of Plate Lysates
A rapid and efficient procedure for purifying bacteriophage h DNA is described. This small-scale purification involves isolation of bacteriophage particles on cesium chloride gradients. Using an Airfuge ultracentrifuge, the centrifugation step can be readily achieved in 90 minutes. The method allows a 1-day purification of up to 12 independent h DNA (20-40 ixg each). The recovered DNA, essentially devoid of R N A and DNA contaminants, is efficiently cut by restriction endonucleases and can serve as starting material for the ligation of DNA fragments in other cloning vehicles.
Bacteriophage h vectors are widely used for the construction of genomic DNA or cDNA libraries. Analysis of isolated recombinant phages involves the purification of their DNA for restriction endonuclease mapping. Large-scale preparation of bacteriophage h DNA can be achieved using several efficient methods [1]. However, isolation of large amounts of bacteriophage h DNA from a great number of clones requires time-consuming and labor-intensive steps. Although powerful methods have been developed for the rapid isolation of plasmid DNA from many samples simultaneously, with a purity allowing analysis by restriction endonuclease digestion [2, 3], the rapid, small-scale isolation of bacteriophage h D N A often represents a more difficult task. Although numerous procedures have been described for the isolation of h DNA [1, 4-6], DNA obtained from phages grown on agarose plates is not cut well by various enzymes. In this study, by using an Airfuge ultracentrifugation step in cesium chloride
From the Laboratoire d'Enzymologie du CNRS, Gif sur Yvette, France. Address reprint requests to: Marc Mirande, Centre National de la Recherche Scientitique, Laboratoire D'Enzymologie, 91190 Gif sur Yvette, France. Received January 22, 1988.
Plate lysates were obtained essentially as described by Maniatis et al. [1], with minor modifications. Escherichiacoli YI090 [7] were grown to saturation, at 37°C, in L medium containing 0.2% maltose. Cells were pelleted by centrifugation and taken up in 10 mM M g S O 4 at 2 A600/ml. Then 100 txl of the bacterial suspension were mixed with 10 fxl of the bacteriophage solution ( 1 0 4 - 1 0 5 pfu). After incubation at 37°C for 20 minutes, 2.7 ml of molten top agarose (Agar Noble, Difco) were added and poured on 95-mm plates freshly prepared with 1.5% agarose in L medium. Plates were made air-tight with Parafilm " M " to retain moisture and were incubated at 42°C. Plaques were allowed to form for 5 - 6 hours until they covered up the surface of the plate. Then 5 ml of SM buffer (50 mM Tris-HCl, pH 7.5,100 mM NaC1, 10 mM M g S O 4, 0.01% gelatin) were added and bacteriophage particles were allowed to diffuse overnight, at 4°C, with gentle shaking. The following procedures were designed for the isolation of bacteriophage DNA starting from two 95-mm plates.
Purification of Bacteriophage Particles The bacteriophage solution in SM buffer ( - 6 - 7 ml) was centrifuged at 10,000g for I0 minutes to remove bacterial debris and residual agarose. To the supernatant, an equal volume of a solution containing 2 M NaC1 and 20% polyethylene glycol 6000 (PEG-6000) was added. Bacteriophage particles were allowed to precipitate for 30 minutes in an ice-water bath. After centrifugation at 10,000g for 10 minutes, the bacteriophage pellet was taken up in 2 ml of TNM buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaC1, 5 mM MgCI2). DNase I and RNase A were added to a final concentration of 1 p~g/ml. Incubation was conducted at 37°C for 30 minutes. After a second precipitation step, at 0°C, for 30 minutes, using 2 ml of the same PEG-6000 solution, bacteriophage particles were
© 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017 0735-0651/88/$03.50
1988, Gene Anal Techn 5:80-82
p e l l e t e d by c e n t r i f u g a t i o n at 10,000g for 10 minutes. The supernatant was thoroughly removed. A further centrifugation at 5,000g for 2 minutes allowed elimination of residual PEG-6000 remaining on the walls of the centrifuge tubes. The residual pellet was taken up with T N M buffer, to a final volume of 210 Ixl. Following addition of 157.5 mg of CsCI and centrifugation at 10,000g for 10 minutes, at 4°C, 190 ~1 of the supernatant were transferred in Ultra-Clear centrifuge tubes (5 × 20 mm). Centrifugation was performed in an Airfuge Ultracentrifuge (Beckman) with the A-100 rotor, at 30 psig (135,000 gay) for 90 minutes, at room temperature.
Isolation of Bacteriophage h D N A The bacteriophage particles obtained after the ultracentrifugation step ( - 3 0 - 4 0 Ixl) were diluted by addition of 500 ixl H20. Following two 500-1xl phenol extractions and two 500-1xl ether extractions, 1 ml of ethanol (at room temperature) was added to the aqueous phase. After a 5-minute centrifugation at 10,000g, at room temperature, the DNA pellet was washed with 500 Ixl ethanol and taken up in 40 txl of TE buffer (10 mM TrisHCI, pH 7.5, 1 mM EDTA). The purified bacteriophage h DNA was kept overnight at 4°C before restriction endonuclease digestion.
fugation step. A f t e r phenol e x t r a c t i o n and e t h a n o l p r e c i p i t a t i o n , the r e c o v e r e d D N A (starting from six plates) was taken up in 120 ~1 of TE buffer. The hSCX23 DNA was purified as described above, except that the bacteriophage suspension was transferred in a single ultracentrifuge tube, starting from two 95-ram plates. Purified DNA was dissolved in 40 p.l of TE buffer. Aliquots of 5 ~1 of hgtll DNA and 10 pJ of hSCX23 DNA were submitted to restriction endonuclease digestions and analyzed on a 1% agarose gel. Electrophoretic patterns, revealed by staining with ethidium bromide, obtained after EcoRI digestion (hSCX23), BamHI, EcoRV, HindIII, KpnI, PvuII or SacI digestions (hgtl 1) are shown in Figure 1. The efficiency of restriction endonuclease digestion is essentially similar to that obFigure 1. Analysis of purified bacteriophage h DNA by re~endonuclease digestions. Bacteriophage h DNA were purified, as described under Materials and Methods, by an Aiffuge centrifugation step in cesium chloride gradients. (A) hSCX23, a hgtll recombinant encoding rat liver aspartyltRNA synthetase; (B-G) wild-type hgtl 1. Purified DNA was digested with restriction endonucleases (Boehringer, Mannheim) as recommended by the supplier and analyzed on a 1% agarose gel. Digestions were carried out with EcoRI (A), SacI (B), PvuII (C), Kpnl (D), HindIII (E), EcoRV (F), and BamHI (G). Left and right lanes (M): size markers, in base pairs.
MA BC DE F G M Results and Discussion Isolation of bacteriophage h DNA was carried out as described in Materials and Methods, using wild-type hgtl 1 [8] and hSCX23, a ~gtl 1 recombinant encoding rat liver aspartyl-tRNA synthetase, a component of the high molecular weight complex containing nine aminoacyl-tRNA synthetases [9], isolated by probing a phage hgtl 1 recombinant rat liver cDNA expression library [10] with antibodies directed to the aminoacyl-tRNA synthetase complex from sheep liver (results to be published). For the purification of hgtl 1 DNA, three dilutions (104-105 PFU) of a h g t l l stock solution were incubated with freshly prepared E. coli Y1090 and poured on 95-mm plates made of 1.5% agarose in L medium (six plates for each dilution). B a c t e r i o p h a g e p a r t i c l e s from p l a t e s showing an almost entirely clear surface were processed further. Following two rounds of PEG precipitation, phages were transferred in three centrifuge tubes and purified by an Airfuge ultracentri-
Uuuu -
2527
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1623
--
849
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441 341 310 214 169 106
© 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017
- j ~-~
7---;
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82 1988, Gene Anal Techn 5:80-82
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tained with bacteriophage h DNA purified by standard large-scale procedures [1]. In addition, k DNA prepared in this way is devoid of low molecular weight RNA contaminants. Thus, small DNA fragments up to 100-150 bp are easily detectable. The purification procedure described in this paper has been routinely employed in our laboratory for more than 6 months. All restriction enzymes used were effective for the digestion of the purified DNA. In addition, the recovered DNA was pure enough for most standard experiments in molecular biology. In particular, the cDNA inserts released after EcoRI digestion of hSCX23 DNA (1627 and 313 bp), or from other kgtll recombinant phages isolated in our laboratory, were efficiently subcloned in M13 [11] or pSP6 [12] vectors. Starting from two 95-mm plates and following exactly the procedure described in Materials and Methods, up to 12 independent k DNA can be purified in a single day, including two consecutive 90-minute ultracentrifugation steps with six samples in each run. Depending on the hgtl 1 recombinants analyzed, approximately 20-40 ~g of pure DNA were routinely obtained. In addition, bacteriophage particles obtained from up to three 95-mm plates can be processed in one single centrifuge tube without dramatically impairing the biologic activity of the recovered DNA fraction. This work was supported in part by grants from the Ecole Polytechnique and from the Centre National de la Recherche
S c i e n t i f i q u e (A.T.P. " O r g a n i s a t i o n et E x p r e s s i o n du G6nome," grant 90.1821). We gratefully acknowledge the help of Daniel Le Corre at the initial stage of this study. The hgtl 1 recombinant rat liver cDNA library was kindly supplied by ]. Schwarzbauer (M.I.T.).
References 1. Maniatis, T., Fritsch, E. E, and Sambrook, J. (1982) in Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 2. Birnboim, H. C., and Doly, J. (1979) Nucleic Acids Res. 7, 1513-1523. 3. Holmes, D. S., and Quigley, M. (1981) Anal. Biochem. 114, 193-197. 4. Davis, R. W., Thomas, M., Cameron, J., St. John, T. P., Scherer, S., and Padgett, R. A. (1980) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.) Vol. 65, Part I, pp. 404-411, Academic Press, New York. 5. Silhavy, T. J., Berman, M. L., and Enquist, L. W. (1984) in Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 6. Kaslow, D. C. (1986) Nucleic Acids Res. 14, 6767. 7. Young, R. A., and Davis, R. W. (1983) Science 222, 778-782. 8. Young, R. A., and Davis, R. W. (1983) Proc. Natl. Acad. Sci. USA 80, 1194-1198. 9. Cirakoglu, B., and Waller, J.-P. (1985) Biochim. Biophys. Acta 829, 173-179. 10. Schwarzbauer, J. E., Tamkun, J. W., Lemischka, I. R., and Hynes, R. O. (1983) Cell 35,421-431. 11. Messing, J. (1983) in Methods in Enzymology (Wu, R., Grossman, L., and Moldave, K., eds.) Vol. 10l, Part C, pp. 20-78, Academic Press, New York. 12. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis. T., Zinn, K., and Green, M. R. (1984) Nucleic Acids Res. 12, 7035-7070.
© 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017
- j ."-. - e'-~
Small-Scale Purification of Bacteriophage h DNA by an Airfuge Centrifugation Step in Cesium Chloride Gradients
gradients, rapid isolation of biologically active, highly purified DNA was accomplished starting from b a c t e r i o p h a g e h g t l l grown on agarose plates. Small amounts of several samples of bacteriophage h DNA can be purified in a single day. The recovered DNA is devoid of RNA and DNA contaminants and is efficiently cut by restriction endonucleases.
MARC MIRANDE, M Y R I A M L A Z A R D , and
Materials and Methods
JEAN-PIERRE WALLER
Preparation of Plate Lysates
A rapid and efficient procedure for purifying bacteriophage h DNA is described. This small-scale purification involves isolation of bacteriophage particles on cesium chloride gradients. Using an Airfuge ultracentrifuge, the centrifugation step can be readily achieved in 90 minutes. The method allows a 1-day purification of up to 12 independent h DNA (20-40 ixg each). The recovered DNA, essentially devoid of R N A and DNA contaminants, is efficiently cut by restriction endonucleases and can serve as starting material for the ligation of DNA fragments in other cloning vehicles.
Bacteriophage h vectors are widely used for the construction of genomic DNA or cDNA libraries. Analysis of isolated recombinant phages involves the purification of their DNA for restriction endonuclease mapping. Large-scale preparation of bacteriophage h DNA can be achieved using several efficient methods [1]. However, isolation of large amounts of bacteriophage h DNA from a great number of clones requires time-consuming and labor-intensive steps. Although powerful methods have been developed for the rapid isolation of plasmid DNA from many samples simultaneously, with a purity allowing analysis by restriction endonuclease digestion [2, 3], the rapid, small-scale isolation of bacteriophage h D N A often represents a more difficult task. Although numerous procedures have been described for the isolation of h DNA [1, 4-6], DNA obtained from phages grown on agarose plates is not cut well by various enzymes. In this study, by using an Airfuge ultracentrifugation step in cesium chloride
From the Laboratoire d'Enzymologie du CNRS, Gif sur Yvette, France. Address reprint requests to: Marc Mirande, Centre National de la Recherche Scientitique, Laboratoire D'Enzymologie, 91190 Gif sur Yvette, France. Received January 22, 1988.
Plate lysates were obtained essentially as described by Maniatis et al. [1], with minor modifications. Escherichiacoli YI090 [7] were grown to saturation, at 37°C, in L medium containing 0.2% maltose. Cells were pelleted by centrifugation and taken up in 10 mM M g S O 4 at 2 A600/ml. Then 100 txl of the bacterial suspension were mixed with 10 fxl of the bacteriophage solution ( 1 0 4 - 1 0 5 pfu). After incubation at 37°C for 20 minutes, 2.7 ml of molten top agarose (Agar Noble, Difco) were added and poured on 95-mm plates freshly prepared with 1.5% agarose in L medium. Plates were made air-tight with Parafilm " M " to retain moisture and were incubated at 42°C. Plaques were allowed to form for 5 - 6 hours until they covered up the surface of the plate. Then 5 ml of SM buffer (50 mM Tris-HCl, pH 7.5,100 mM NaC1, 10 mM M g S O 4, 0.01% gelatin) were added and bacteriophage particles were allowed to diffuse overnight, at 4°C, with gentle shaking. The following procedures were designed for the isolation of bacteriophage DNA starting from two 95-mm plates.
Purification of Bacteriophage Particles The bacteriophage solution in SM buffer ( - 6 - 7 ml) was centrifuged at 10,000g for I0 minutes to remove bacterial debris and residual agarose. To the supernatant, an equal volume of a solution containing 2 M NaC1 and 20% polyethylene glycol 6000 (PEG-6000) was added. Bacteriophage particles were allowed to precipitate for 30 minutes in an ice-water bath. After centrifugation at 10,000g for 10 minutes, the bacteriophage pellet was taken up in 2 ml of TNM buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaC1, 5 mM MgCI2). DNase I and RNase A were added to a final concentration of 1 p~g/ml. Incubation was conducted at 37°C for 30 minutes. After a second precipitation step, at 0°C, for 30 minutes, using 2 ml of the same PEG-6000 solution, bacteriophage particles were
© 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017 0735-0651/88/$03.50
1988, Gene Anal Techn 5:80-82
p e l l e t e d by c e n t r i f u g a t i o n at 10,000g for 10 minutes. The supernatant was thoroughly removed. A further centrifugation at 5,000g for 2 minutes allowed elimination of residual PEG-6000 remaining on the walls of the centrifuge tubes. The residual pellet was taken up with T N M buffer, to a final volume of 210 Ixl. Following addition of 157.5 mg of CsCI and centrifugation at 10,000g for 10 minutes, at 4°C, 190 ~1 of the supernatant were transferred in Ultra-Clear centrifuge tubes (5 × 20 mm). Centrifugation was performed in an Airfuge Ultracentrifuge (Beckman) with the A-100 rotor, at 30 psig (135,000 gay) for 90 minutes, at room temperature.
Isolation of Bacteriophage h D N A The bacteriophage particles obtained after the ultracentrifugation step ( - 3 0 - 4 0 Ixl) were diluted by addition of 500 ixl H20. Following two 500-1xl phenol extractions and two 500-1xl ether extractions, 1 ml of ethanol (at room temperature) was added to the aqueous phase. After a 5-minute centrifugation at 10,000g, at room temperature, the DNA pellet was washed with 500 Ixl ethanol and taken up in 40 txl of TE buffer (10 mM TrisHCI, pH 7.5, 1 mM EDTA). The purified bacteriophage h DNA was kept overnight at 4°C before restriction endonuclease digestion.
fugation step. A f t e r phenol e x t r a c t i o n and e t h a n o l p r e c i p i t a t i o n , the r e c o v e r e d D N A (starting from six plates) was taken up in 120 ~1 of TE buffer. The hSCX23 DNA was purified as described above, except that the bacteriophage suspension was transferred in a single ultracentrifuge tube, starting from two 95-ram plates. Purified DNA was dissolved in 40 p.l of TE buffer. Aliquots of 5 ~1 of hgtll DNA and 10 pJ of hSCX23 DNA were submitted to restriction endonuclease digestions and analyzed on a 1% agarose gel. Electrophoretic patterns, revealed by staining with ethidium bromide, obtained after EcoRI digestion (hSCX23), BamHI, EcoRV, HindIII, KpnI, PvuII or SacI digestions (hgtl 1) are shown in Figure 1. The efficiency of restriction endonuclease digestion is essentially similar to that obFigure 1. Analysis of purified bacteriophage h DNA by re~endonuclease digestions. Bacteriophage h DNA were purified, as described under Materials and Methods, by an Aiffuge centrifugation step in cesium chloride gradients. (A) hSCX23, a hgtll recombinant encoding rat liver aspartyltRNA synthetase; (B-G) wild-type hgtl 1. Purified DNA was digested with restriction endonucleases (Boehringer, Mannheim) as recommended by the supplier and analyzed on a 1% agarose gel. Digestions were carried out with EcoRI (A), SacI (B), PvuII (C), Kpnl (D), HindIII (E), EcoRV (F), and BamHI (G). Left and right lanes (M): size markers, in base pairs.
MA BC DE F G M Results and Discussion Isolation of bacteriophage h DNA was carried out as described in Materials and Methods, using wild-type hgtl 1 [8] and hSCX23, a ~gtl 1 recombinant encoding rat liver aspartyl-tRNA synthetase, a component of the high molecular weight complex containing nine aminoacyl-tRNA synthetases [9], isolated by probing a phage hgtl 1 recombinant rat liver cDNA expression library [10] with antibodies directed to the aminoacyl-tRNA synthetase complex from sheep liver (results to be published). For the purification of hgtl 1 DNA, three dilutions (104-105 PFU) of a h g t l l stock solution were incubated with freshly prepared E. coli Y1090 and poured on 95-mm plates made of 1.5% agarose in L medium (six plates for each dilution). B a c t e r i o p h a g e p a r t i c l e s from p l a t e s showing an almost entirely clear surface were processed further. Following two rounds of PEG precipitation, phages were transferred in three centrifuge tubes and purified by an Airfuge ultracentri-
Uuuu -
2527
--
1623
--
849
-
441 341 310 214 169 106
© 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017
- j ~-~
7---;
-___---
-j
82 1988, Gene Anal Techn 5:80-82
..---j
....-
..._-----
tained with bacteriophage h DNA purified by standard large-scale procedures [1]. In addition, k DNA prepared in this way is devoid of low molecular weight RNA contaminants. Thus, small DNA fragments up to 100-150 bp are easily detectable. The purification procedure described in this paper has been routinely employed in our laboratory for more than 6 months. All restriction enzymes used were effective for the digestion of the purified DNA. In addition, the recovered DNA was pure enough for most standard experiments in molecular biology. In particular, the cDNA inserts released after EcoRI digestion of hSCX23 DNA (1627 and 313 bp), or from other kgtll recombinant phages isolated in our laboratory, were efficiently subcloned in M13 [11] or pSP6 [12] vectors. Starting from two 95-mm plates and following exactly the procedure described in Materials and Methods, up to 12 independent k DNA can be purified in a single day, including two consecutive 90-minute ultracentrifugation steps with six samples in each run. Depending on the hgtl 1 recombinants analyzed, approximately 20-40 ~g of pure DNA were routinely obtained. In addition, bacteriophage particles obtained from up to three 95-mm plates can be processed in one single centrifuge tube without dramatically impairing the biologic activity of the recovered DNA fraction. This work was supported in part by grants from the Ecole Polytechnique and from the Centre National de la Recherche
S c i e n t i f i q u e (A.T.P. " O r g a n i s a t i o n et E x p r e s s i o n du G6nome," grant 90.1821). We gratefully acknowledge the help of Daniel Le Corre at the initial stage of this study. The hgtl 1 recombinant rat liver cDNA library was kindly supplied by ]. Schwarzbauer (M.I.T.).
References 1. Maniatis, T., Fritsch, E. E, and Sambrook, J. (1982) in Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 2. Birnboim, H. C., and Doly, J. (1979) Nucleic Acids Res. 7, 1513-1523. 3. Holmes, D. S., and Quigley, M. (1981) Anal. Biochem. 114, 193-197. 4. Davis, R. W., Thomas, M., Cameron, J., St. John, T. P., Scherer, S., and Padgett, R. A. (1980) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.) Vol. 65, Part I, pp. 404-411, Academic Press, New York. 5. Silhavy, T. J., Berman, M. L., and Enquist, L. W. (1984) in Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 6. Kaslow, D. C. (1986) Nucleic Acids Res. 14, 6767. 7. Young, R. A., and Davis, R. W. (1983) Science 222, 778-782. 8. Young, R. A., and Davis, R. W. (1983) Proc. Natl. Acad. Sci. USA 80, 1194-1198. 9. Cirakoglu, B., and Waller, J.-P. (1985) Biochim. Biophys. Acta 829, 173-179. 10. Schwarzbauer, J. E., Tamkun, J. W., Lemischka, I. R., and Hynes, R. O. (1983) Cell 35,421-431. 11. Messing, J. (1983) in Methods in Enzymology (Wu, R., Grossman, L., and Moldave, K., eds.) Vol. 10l, Part C, pp. 20-78, Academic Press, New York. 12. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis. T., Zinn, K., and Green, M. R. (1984) Nucleic Acids Res. 12, 7035-7070.
© 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017