ANALYTICAL
BIOCHEMISTRY
195,
Ill-115
(19%)
A Method for Eluting DNA in a Wide Range of Molecular Weights from Agarose Gels Giovanni
Maria
Duro,* Vincenzo Izzo,* Rainer Barbieri,? Assunta Costa,* and Giovanni Giudice**t
Maria
Cantone,?
*Istituto di Biologia dell0 Sviluppo, C.N.R. Via Archirafi, 20-90123 Palermo, Italy; and TDipartimento di Biologia Cellulure e dell0 Sviluppo Via Archiraji, 22-90123 Palermo, Italy
Received
July
6, 1990
We have developed a simple and rapid method for recovering DNAs of a wide range of molecular weights from agarose gels. A DNA-containing gel slice is placed on a Parafilm sheet in the center of a circular (positive) electrode and covered with a drop of buffer, while a linear (negative) electrode is placed on the top of the gel and driven about 1 mm into the gel itself. When a continuous current is applied, the DNA migrates into the buffer toward the circular electrode. We have obtained almost total recovery of DNAs up to 10 kb in size. Our method may also be used, under appropriate conditions, for higher molecular weight DNAs. The yield and all the biological assays performed on the DNAs obtained by our method recommend it for routine laboratory use. 0 1991 Academic Press, Inc.
Agarose gel electrophoresis is a powerful technique for separating nucleic acids on the basis of different sizes and conformations (l-3). One of the limiting steps in the usual laboratory praxis is the recovery of separated DNA fragments from the gel itself in order to use them for further studies. In the past 10 years many chemical and physical techniques for obtaining pure DNA fragments from agarose gels (4-19) based on chemical release of DNA from the gel or recovering by electroelution have been developed. But many of these methods require long times, costly apparatus, or chemical and physical manipulations that reduce the yield or cause damage to the DNA itself. MATERIALS
AND
METHODS
Principle of the Method Our method makes use of two platinum electrodes (1 mm thick), one (positive) circular and the other (nega0003-2697/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
tive) linear, perpendicular to the plane of the first. The DNA-containing agarose gel slice is placed on a Parafilm sheet, and the circular electrode (whose diameter can be varied as a function of the gel slice size, in a range of 1 to 2 cm) is placed around it. The complex is then covered with a drop of 1X TBE (0.3-1.0 ml), the volume of which depends on the size of the gel slice and on the diameter of the circular electrode, so as to surround the gel slice and to adhere to the inner edge of the circular electrode. The linear electrode is placed on top of the gel and driven about 1 mm into the gel itself (Fig. la). When the gel slices are very thin, it is sufficient only for the cathode to firmly touch the top of the gel. El&ion Procedure and DNA Purification When a 25-V continuous current is applied to the system, the DNA in the gel rapidly and radially migrates toward the circular electrode in the surrounding buffer, a process that can be followed under a uv lamp. After DNA migration the gel slice is removed and placed on a transilluminator (wavelength 312 nm) to check the complete elution of the DNA from the gel. Then the buffer (a maximum of 1 ml, one of the advantages of this method) is collected with a pipet and the DNA is extracted with 1 vol of chloroform. Sodium acetate is added to a final concentration of 0.3 M, the mixture is precipitated for 15 min at 0°C with 2 vol of cold ethanol, and the DNA is collected by centrifugation at 12,000g for 15 min at 4°C. Pure DNA can be obtained in less than 1 h by this method. For easier use of our system we have built the very simple apparatus shown in Fig. lb. After each round of elution the electrodes were cleaned by flaming with a Bunsen. The timing of the entire elution depends on the molecular weight and the concentration of the eluted DNA and on the agarose concentration. The complete DNA 111
Inc. reserved.
112
DURO
ET
AL.
a FIG. 1. (a) Schematic view of the elution system. A DNA-containing gel slice is placed on a TBE drop and surrounded by a circular (+) electrode; the linear (-) electrode is positioned on top of the gel slice. The entire system is placed on a Parafilm sheet. (b) A photograph of our elution apparatus. The circular electrode is fixed to the base of the apparatus while the linear electrode is free to move vertically.
migration can be followed by a uv lamp (Longlife filter, Spectroline) and the time for complete elution, under the conditions described above, varies from 1 to 4 min. We did not detect significant variations in the pH of the buffer or the temperature during the process, nor interference due to the ethidium bromide staining.
Preparative
Gel Electrophoresis
We have used preparative gels ranging from 0.7 to 1.5% (w/v) agarose (International Biotechnologies Inc., Molecular Biology grade, ultra pure reagent) in TBE buffer (89 mM Tris pH 8.3, 89 mM boric acid, 8 mM EDTA) with 0.5 pg/ml of ethidium bromide (added when the gel solution was at about 50°C). The samples were run on horizontal submarine gels with constant current in 1X TBE buffer. A razor blade was used to excise the band of interest from the gel. The dimension of the gel slices varies as a function of the comb tooth used in the electrophoresis; in our experiments comb teeth varying from 5 to 15 mm were routinely used, with a gel thickness ranging from 2 to 5 mm.
Nucleic Acids Manipulations Conditions
and Hybridization
All the digestion assays with restriction enzymes were carried out according to the manufacturer’s specifications. The DNAs used for recovery experiments were 32Pend-labeled (20). DNA probes used for hybridization experiments were 32P-labeled by the random primer method (21). The T4 DNA ligase reactions were performed according to the manufacturer’s specifications and the transformation experiments were performed as described by Sambrook et al. (20).
Southern blot hybridizations were performed as described by Sambrook et al. (20), and the denaturated DNAs were transferred onto a Hybond membrane (Hybond N, Amersham) in 50 IIIM sodium phosphate, pH 7.0. The DNAs were denatured in 1% agarose gels in EB buffer (50 IIIM boric acid, 5 InM sodium borate, 10 mM sodium sulfate, 1 mM EDTA) in the presence of 7.5 mM methylmercuric hydroxide (Alfa Ventron) according to Sambrook et al. (20). After electrophoresis in EB buffer, the gel was neutralized in 0.5 M ammonium acetate and blotted onto a Hybond membrane in 50 mM sodium phosphate, pH 7.0, according to Sambrook et al. (20).
RESULTS
DNA molecules ranging from a few hundred base pairs (bp) to about 10 kilobases (kb) were quickly eluted by the method described above. The yield of eluted DNA was estimated by measuring the radioactivity of 32P end-labeled DNAs before and after elution. The data reported in Table 1 indicate that more than 90% of all the DNAs in a wide range of molecular weights were recovered. The yield of the same DNAs measured by gel staining after precipitation was the same. The electrophoretic mobility of different DNAs recovered from agarose gels with our procedure (indicated as “eluted”) is shown in Fig. 2 in comparison with the same DNAs that had not been subjected to any elution (indicated as “noneluted”). The results clearly indicate that our elution procedure has not modified the electrophoretie properties of the eluted DNAs. Furthermore we have carried out a series of experiments aimed at demonstrating the suitability of these
DNA TABLE
Percentage
DNA
ELUTION
FROM
AGAROSE
kb
1
Recovery of the 32P End-Labeled according to the Size
DNA
Percentage of cpm eluted
size (kb) 0.4 2.6 6.2 8.3 9.8 48 A B
95 94 92 92 91
f + + + +
5 5 5 5 5
21+ 5 64 + 5
Note. A: Recovery with the standard the modified procedure. Five recovery for each DNA size.
procedure. experiments
B: Recovery with were carried out
a FIG. lanes
eluted DNAs for molecular biology. Different eluted and noneluted DNAs were digested under the same conditions with a series of restriction enzymes and, as shown in Figs. 2 and 4, the restriction patterns of the eluted DNAs do not show any change from those of the same DNAs before elution. We also tested the suitability of eluted DNAs for ligation reactions: a 6.2kb plasmid (our clone pRr 19/B3, obtained by cloning the 3.2-kb EcoRI-BumHI region of a sea urchin ribosomal DNA spacer in a Bluescript vector) was linearized with EcoRI. The EcoRI-digested clone was electrophoresed, eluted, and used for T4 DNA ligase reactions together with the EcoRI-linearized noneluted one. The religated clones were used to transform Escherichia coli XLl-Blue strains. The transforming efficiency, obtained in a wide series of experiments, using
ab
113
GELS
c
d
e
f kb
-6.2 -3 -2 1.2
FIG. 2. One percent agarose gel electrophoresis of equal amounts of noneluted (lane a) and eluted (lane b) EcoRI-linearized pRr19/B3 DNA. A noneluted EcoRI-P.&-digested pRr19/B3 (lane c!) is compared with each of its single eluted bands (lanes d, e, and f’).
b
c
d
3. Denaturing gel hybridization of the same DNAs used in c, d, and f of Fig. 2 using 32P-labeled pRr19/B3 as a probe.
eluted and noneluted religated DNAs was the same (about 5 x 10’ transformants1p.g of DNA). We have also carried out Southern blot hybridization experiments using eluted DNAs (uncut X DNA and EcoRI- and HindIII-restricted X DNA; see below) to demonstrate the complete suitability of such DNAs for denaturation and hybridization to a homologous probe. The result of one such experiment is shown in Fig. 4, where no difference can be seen between the hybridization patterns of the noneluted and the eluted DNAs. Finally, to test the possibility that during the elution process the DNA could be subjected to single-strand nicks, we carried out gel denaturation experiments using methylmercuric hydroxide denaturing gels in which eluted and noneluted DNAs were compared. An EcoRIP&I-digested, eluted pRr19/B3 plasmid was electrophoresed in a 1% agarose gel in EB buffer with 7.5 mM methylmercuric hydroxide, transferred onto a Hybond membrane, and hybridized with a 32P random-primerlabeled pRr19/B3. The result of this experiment, shown in Fig. 3, demonstrates that the eluted DNA has not undergone any variation in migration pattern under denaturing conditions and therefore has not been singlestrand nicked during the elution process. For larger DNAs, i.e., greater than 10 kb, we observed a much lower recovery (about 20%) after the standard elution time of about 4 min. Longer elution times or changes in the field strength did not cause any substantial improvement in the yield. We therefore modified the procedure by changing the elution buffer every 4 min with a pipet when DNAs of sizes longer than 10 kb were involved. This modification allowed us to recover about 60% of a 48-kb-long DNA, such as the X phage. Southern blot experiments show also that DNA eluted with our method retains its suitability for biological use as shown in Fig. 4 where the restriction pattern of this
114
DURO
a
b
c
d
e
f
FIG. 4. Southern blot hybridization of the noneluted (a) and eluted (b) uncut X DNA, EcoRI noneluted (c) and eluted (d) X DNA, and Hind111 noneluted (e) and “eluted” (f) X DNA, using “P-labeled X DNA as a probe. (The lower molecular weight bands in lanes c, d, e, and f were out of the gel.)
48-kb phage DNA after elution is compared with its pattern before elution.
DISCUSSION
In the past years many methods for recovering DNA fragments from agarose gels have been described. In our opinion an efficient and reliable method must satisfy the following conditions: (a) it must allow a very good quantitative and qualitative recovery of the material; (b) it should be rapid and simple to use; (c) it must involve minimal handling of the material; (d) it should satisfy all the above conditions at the lowest cost. We have developed a method that satisfies all the above conditions together with providing capability of following the elution under a uv lamp and offering the great advantage of obtaining DNA in small volumes. Due to the peculiarity of our system, it is almost impossible to lose the DNA being recovered, and the method may be used when low DNA concentrations are being employed. We have carried out a series of elution experiments on a wide range of molecular weight DNAs using different buffers, times, and voltages: the optimal conditions we found were 25 V in 1X TBE buffer in a maximum time of 4 min. Under these conditions all the DNAs tested in a molecular weight ranging from a few hundred base pairs to 10 kb can be recovered with the very high yield reported in Table 1. When the elution was carried out for longer times, we noted a progressive decrease in the yield of recovered DNAs. DNA longer than 10 kb requires more time than a smaller DNA to be eluted from the gel, so that a standard elution time (about 4 min) is sufficient to transfer only part of it. Longer elution times (about 15 min for 5 rg of X DNA in 0.8% w/v
ET
AL.
agarose gel) are necessary to remove all of these DNAs from the gel. Under these conditions, however, only 20% of the DNA was recovered in the buffer. What causes this DNA loss? Although part of the eluted DNA was found bound to the anode, this amount did not justify the low recovery, but raised the possibility that the DNA was completely degraded by anodic oxidation while bound to the anode. This possibility is also supported by the observation that the DNA concentration in the buffer was observed to decrease with elution time. We therefore attempted to overcome this problem by substituting the TBE buffer every 4 min during the elution of the very high molecular weight DNAs. This modification of the procedure resulted in a higher volume of DNA solution but allowed us to obtain a satisfactory DNA recovery (about 60%). Although it is theoretically possible to continuously remove the elution buffer with a peristaltic pump, we have found it much simpler to use a pipet to withdraw and replace the elution buffer every 4 min. The possibility that pulse-field gel electrophoresis may increase the recovery of DNA of sizes larger than 10 kb remains to be investigated. Although other methods allow a qualitative and quantitative DNA recovery similar to ours, e.g., electroelution into dialysis bags (20), our method is much more rapid (1 h to obtain pure DNAs) and evidently simpler, and offers the advantage of enabling the operator to directly follow DNA elution under the uv lamp. All the biological assays performed on all the DNAs used in our experiments showed the validity and reliability of our method. This fact, together with its simplicity and rapidity, recommends this method for routine laboratory use.
ACKNOWLEDGMENTS This work was supported in part by funds of the “Progetto finalizzato Biotecnologie e Biostrumentazione” of the Italian National Research Council, and by funds of the Italian MURST (40 and 60%). We are indebted to Dr. Alexander A. Chisholm for revising the English form. Patent No. 91A000249.
REFERENCES 1. Thorne, 2. Thorne,
H. V. (1966) H. V. (1967)
Virology 29, 234-241. J. Mol. Biol. 24,203-211.
3. Takahashi, M., Ogino, T., and Baba, K. (1969) Biochem. Biophys. Acta 174,183-187. 4. Tabak, H. F., and Flavell, R. A. (1978) Nucleic Acids Res. 5,23212332. 5. Volgelstein, B., and Gillespie, D. (1979) hoc. Natl. USA 76,615619. 6. Chen, C. W., and Thomas, C. A. (1980) Anal. Biochem. 341.
Acad.
Sci.
101,339-
DNA 7. Saha, B. K., Strelow, S., and Schlessinger, Biophys. Methods 7,277-284. 8. Wende,
M.,
Dorbic,
T., and Wittig,
ELUTION
D. (1983)
B. (1985)
FROM
J. Biochem.
15. Zassenhaus, Biochem.
Nucleic
Acids
Res.
J. (1986)
Anal.
Bio-
13,9043. 9. McEnery, M. W., Angus, them. 156,72-75. 10. Volgelstein,
B. (1987)
Anal.
W. C., and Moss,
11. Southern, E. (1979) in Methods Vol. 68, p. 178, Academic Press, 12. MC Donell, Mol. Biol. 13. Veislander,
M.
W., Simon,
160, 115-l
Biochem.
M.
18.
in Enzymology New York. N., and
Studier,
(Wu,
R., Ed.),
F. W. (1977)
J.
110, 119-146. L. (1979)
14. Dretzen, G., Bellard, (1981) Anal. Biochem.
Anal. M.,
Biochem.
98.305-309.
Sassone-Corsi,
112, 295-298.
P., and
Chambon,
AGAROSE
P.
115
GELS P. H., Butow,
R. A., and Hannon,
Y. P. (1982)
Anal.
125,125-130.
16. Tautz, D., and Renz, M. (1983) Anal. Biochem. 132,14-19. 17. Ofverstedt, L., Hammarstrom, K., Balgobin, N., Hjerten, N., Pettersson, U., and Chattopadhyaya, J. (1984) Biochem. Biophys. Actn 782,2321-2332. 18. Orban, L., Hahn, 167-171. 19. Pollman, 12-17.
E., and Chrambach,
M. J., and Zuccarelli,
A. (1988)
A. J. (1989)
Ekctrophoresis
Anal.
Biochem.
9,
181,
20. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 21. Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13.
BIOCHEMISTRY
195,
Ill-115
(19%)
A Method for Eluting DNA in a Wide Range of Molecular Weights from Agarose Gels Giovanni
Maria
Duro,* Vincenzo Izzo,* Rainer Barbieri,? Assunta Costa,* and Giovanni Giudice**t
Maria
Cantone,?
*Istituto di Biologia dell0 Sviluppo, C.N.R. Via Archirafi, 20-90123 Palermo, Italy; and TDipartimento di Biologia Cellulure e dell0 Sviluppo Via Archiraji, 22-90123 Palermo, Italy
Received
July
6, 1990
We have developed a simple and rapid method for recovering DNAs of a wide range of molecular weights from agarose gels. A DNA-containing gel slice is placed on a Parafilm sheet in the center of a circular (positive) electrode and covered with a drop of buffer, while a linear (negative) electrode is placed on the top of the gel and driven about 1 mm into the gel itself. When a continuous current is applied, the DNA migrates into the buffer toward the circular electrode. We have obtained almost total recovery of DNAs up to 10 kb in size. Our method may also be used, under appropriate conditions, for higher molecular weight DNAs. The yield and all the biological assays performed on the DNAs obtained by our method recommend it for routine laboratory use. 0 1991 Academic Press, Inc.
Agarose gel electrophoresis is a powerful technique for separating nucleic acids on the basis of different sizes and conformations (l-3). One of the limiting steps in the usual laboratory praxis is the recovery of separated DNA fragments from the gel itself in order to use them for further studies. In the past 10 years many chemical and physical techniques for obtaining pure DNA fragments from agarose gels (4-19) based on chemical release of DNA from the gel or recovering by electroelution have been developed. But many of these methods require long times, costly apparatus, or chemical and physical manipulations that reduce the yield or cause damage to the DNA itself. MATERIALS
AND
METHODS
Principle of the Method Our method makes use of two platinum electrodes (1 mm thick), one (positive) circular and the other (nega0003-2697/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
tive) linear, perpendicular to the plane of the first. The DNA-containing agarose gel slice is placed on a Parafilm sheet, and the circular electrode (whose diameter can be varied as a function of the gel slice size, in a range of 1 to 2 cm) is placed around it. The complex is then covered with a drop of 1X TBE (0.3-1.0 ml), the volume of which depends on the size of the gel slice and on the diameter of the circular electrode, so as to surround the gel slice and to adhere to the inner edge of the circular electrode. The linear electrode is placed on top of the gel and driven about 1 mm into the gel itself (Fig. la). When the gel slices are very thin, it is sufficient only for the cathode to firmly touch the top of the gel. El&ion Procedure and DNA Purification When a 25-V continuous current is applied to the system, the DNA in the gel rapidly and radially migrates toward the circular electrode in the surrounding buffer, a process that can be followed under a uv lamp. After DNA migration the gel slice is removed and placed on a transilluminator (wavelength 312 nm) to check the complete elution of the DNA from the gel. Then the buffer (a maximum of 1 ml, one of the advantages of this method) is collected with a pipet and the DNA is extracted with 1 vol of chloroform. Sodium acetate is added to a final concentration of 0.3 M, the mixture is precipitated for 15 min at 0°C with 2 vol of cold ethanol, and the DNA is collected by centrifugation at 12,000g for 15 min at 4°C. Pure DNA can be obtained in less than 1 h by this method. For easier use of our system we have built the very simple apparatus shown in Fig. lb. After each round of elution the electrodes were cleaned by flaming with a Bunsen. The timing of the entire elution depends on the molecular weight and the concentration of the eluted DNA and on the agarose concentration. The complete DNA 111
Inc. reserved.
112
DURO
ET
AL.
a FIG. 1. (a) Schematic view of the elution system. A DNA-containing gel slice is placed on a TBE drop and surrounded by a circular (+) electrode; the linear (-) electrode is positioned on top of the gel slice. The entire system is placed on a Parafilm sheet. (b) A photograph of our elution apparatus. The circular electrode is fixed to the base of the apparatus while the linear electrode is free to move vertically.
migration can be followed by a uv lamp (Longlife filter, Spectroline) and the time for complete elution, under the conditions described above, varies from 1 to 4 min. We did not detect significant variations in the pH of the buffer or the temperature during the process, nor interference due to the ethidium bromide staining.
Preparative
Gel Electrophoresis
We have used preparative gels ranging from 0.7 to 1.5% (w/v) agarose (International Biotechnologies Inc., Molecular Biology grade, ultra pure reagent) in TBE buffer (89 mM Tris pH 8.3, 89 mM boric acid, 8 mM EDTA) with 0.5 pg/ml of ethidium bromide (added when the gel solution was at about 50°C). The samples were run on horizontal submarine gels with constant current in 1X TBE buffer. A razor blade was used to excise the band of interest from the gel. The dimension of the gel slices varies as a function of the comb tooth used in the electrophoresis; in our experiments comb teeth varying from 5 to 15 mm were routinely used, with a gel thickness ranging from 2 to 5 mm.
Nucleic Acids Manipulations Conditions
and Hybridization
All the digestion assays with restriction enzymes were carried out according to the manufacturer’s specifications. The DNAs used for recovery experiments were 32Pend-labeled (20). DNA probes used for hybridization experiments were 32P-labeled by the random primer method (21). The T4 DNA ligase reactions were performed according to the manufacturer’s specifications and the transformation experiments were performed as described by Sambrook et al. (20).
Southern blot hybridizations were performed as described by Sambrook et al. (20), and the denaturated DNAs were transferred onto a Hybond membrane (Hybond N, Amersham) in 50 IIIM sodium phosphate, pH 7.0. The DNAs were denatured in 1% agarose gels in EB buffer (50 IIIM boric acid, 5 InM sodium borate, 10 mM sodium sulfate, 1 mM EDTA) in the presence of 7.5 mM methylmercuric hydroxide (Alfa Ventron) according to Sambrook et al. (20). After electrophoresis in EB buffer, the gel was neutralized in 0.5 M ammonium acetate and blotted onto a Hybond membrane in 50 mM sodium phosphate, pH 7.0, according to Sambrook et al. (20).
RESULTS
DNA molecules ranging from a few hundred base pairs (bp) to about 10 kilobases (kb) were quickly eluted by the method described above. The yield of eluted DNA was estimated by measuring the radioactivity of 32P end-labeled DNAs before and after elution. The data reported in Table 1 indicate that more than 90% of all the DNAs in a wide range of molecular weights were recovered. The yield of the same DNAs measured by gel staining after precipitation was the same. The electrophoretic mobility of different DNAs recovered from agarose gels with our procedure (indicated as “eluted”) is shown in Fig. 2 in comparison with the same DNAs that had not been subjected to any elution (indicated as “noneluted”). The results clearly indicate that our elution procedure has not modified the electrophoretie properties of the eluted DNAs. Furthermore we have carried out a series of experiments aimed at demonstrating the suitability of these
DNA TABLE
Percentage
DNA
ELUTION
FROM
AGAROSE
kb
1
Recovery of the 32P End-Labeled according to the Size
DNA
Percentage of cpm eluted
size (kb) 0.4 2.6 6.2 8.3 9.8 48 A B
95 94 92 92 91
f + + + +
5 5 5 5 5
21+ 5 64 + 5
Note. A: Recovery with the standard the modified procedure. Five recovery for each DNA size.
procedure. experiments
B: Recovery with were carried out
a FIG. lanes
eluted DNAs for molecular biology. Different eluted and noneluted DNAs were digested under the same conditions with a series of restriction enzymes and, as shown in Figs. 2 and 4, the restriction patterns of the eluted DNAs do not show any change from those of the same DNAs before elution. We also tested the suitability of eluted DNAs for ligation reactions: a 6.2kb plasmid (our clone pRr 19/B3, obtained by cloning the 3.2-kb EcoRI-BumHI region of a sea urchin ribosomal DNA spacer in a Bluescript vector) was linearized with EcoRI. The EcoRI-digested clone was electrophoresed, eluted, and used for T4 DNA ligase reactions together with the EcoRI-linearized noneluted one. The religated clones were used to transform Escherichia coli XLl-Blue strains. The transforming efficiency, obtained in a wide series of experiments, using
ab
113
GELS
c
d
e
f kb
-6.2 -3 -2 1.2
FIG. 2. One percent agarose gel electrophoresis of equal amounts of noneluted (lane a) and eluted (lane b) EcoRI-linearized pRr19/B3 DNA. A noneluted EcoRI-P.&-digested pRr19/B3 (lane c!) is compared with each of its single eluted bands (lanes d, e, and f’).
b
c
d
3. Denaturing gel hybridization of the same DNAs used in c, d, and f of Fig. 2 using 32P-labeled pRr19/B3 as a probe.
eluted and noneluted religated DNAs was the same (about 5 x 10’ transformants1p.g of DNA). We have also carried out Southern blot hybridization experiments using eluted DNAs (uncut X DNA and EcoRI- and HindIII-restricted X DNA; see below) to demonstrate the complete suitability of such DNAs for denaturation and hybridization to a homologous probe. The result of one such experiment is shown in Fig. 4, where no difference can be seen between the hybridization patterns of the noneluted and the eluted DNAs. Finally, to test the possibility that during the elution process the DNA could be subjected to single-strand nicks, we carried out gel denaturation experiments using methylmercuric hydroxide denaturing gels in which eluted and noneluted DNAs were compared. An EcoRIP&I-digested, eluted pRr19/B3 plasmid was electrophoresed in a 1% agarose gel in EB buffer with 7.5 mM methylmercuric hydroxide, transferred onto a Hybond membrane, and hybridized with a 32P random-primerlabeled pRr19/B3. The result of this experiment, shown in Fig. 3, demonstrates that the eluted DNA has not undergone any variation in migration pattern under denaturing conditions and therefore has not been singlestrand nicked during the elution process. For larger DNAs, i.e., greater than 10 kb, we observed a much lower recovery (about 20%) after the standard elution time of about 4 min. Longer elution times or changes in the field strength did not cause any substantial improvement in the yield. We therefore modified the procedure by changing the elution buffer every 4 min with a pipet when DNAs of sizes longer than 10 kb were involved. This modification allowed us to recover about 60% of a 48-kb-long DNA, such as the X phage. Southern blot experiments show also that DNA eluted with our method retains its suitability for biological use as shown in Fig. 4 where the restriction pattern of this
114
DURO
a
b
c
d
e
f
FIG. 4. Southern blot hybridization of the noneluted (a) and eluted (b) uncut X DNA, EcoRI noneluted (c) and eluted (d) X DNA, and Hind111 noneluted (e) and “eluted” (f) X DNA, using “P-labeled X DNA as a probe. (The lower molecular weight bands in lanes c, d, e, and f were out of the gel.)
48-kb phage DNA after elution is compared with its pattern before elution.
DISCUSSION
In the past years many methods for recovering DNA fragments from agarose gels have been described. In our opinion an efficient and reliable method must satisfy the following conditions: (a) it must allow a very good quantitative and qualitative recovery of the material; (b) it should be rapid and simple to use; (c) it must involve minimal handling of the material; (d) it should satisfy all the above conditions at the lowest cost. We have developed a method that satisfies all the above conditions together with providing capability of following the elution under a uv lamp and offering the great advantage of obtaining DNA in small volumes. Due to the peculiarity of our system, it is almost impossible to lose the DNA being recovered, and the method may be used when low DNA concentrations are being employed. We have carried out a series of elution experiments on a wide range of molecular weight DNAs using different buffers, times, and voltages: the optimal conditions we found were 25 V in 1X TBE buffer in a maximum time of 4 min. Under these conditions all the DNAs tested in a molecular weight ranging from a few hundred base pairs to 10 kb can be recovered with the very high yield reported in Table 1. When the elution was carried out for longer times, we noted a progressive decrease in the yield of recovered DNAs. DNA longer than 10 kb requires more time than a smaller DNA to be eluted from the gel, so that a standard elution time (about 4 min) is sufficient to transfer only part of it. Longer elution times (about 15 min for 5 rg of X DNA in 0.8% w/v
ET
AL.
agarose gel) are necessary to remove all of these DNAs from the gel. Under these conditions, however, only 20% of the DNA was recovered in the buffer. What causes this DNA loss? Although part of the eluted DNA was found bound to the anode, this amount did not justify the low recovery, but raised the possibility that the DNA was completely degraded by anodic oxidation while bound to the anode. This possibility is also supported by the observation that the DNA concentration in the buffer was observed to decrease with elution time. We therefore attempted to overcome this problem by substituting the TBE buffer every 4 min during the elution of the very high molecular weight DNAs. This modification of the procedure resulted in a higher volume of DNA solution but allowed us to obtain a satisfactory DNA recovery (about 60%). Although it is theoretically possible to continuously remove the elution buffer with a peristaltic pump, we have found it much simpler to use a pipet to withdraw and replace the elution buffer every 4 min. The possibility that pulse-field gel electrophoresis may increase the recovery of DNA of sizes larger than 10 kb remains to be investigated. Although other methods allow a qualitative and quantitative DNA recovery similar to ours, e.g., electroelution into dialysis bags (20), our method is much more rapid (1 h to obtain pure DNAs) and evidently simpler, and offers the advantage of enabling the operator to directly follow DNA elution under the uv lamp. All the biological assays performed on all the DNAs used in our experiments showed the validity and reliability of our method. This fact, together with its simplicity and rapidity, recommends this method for routine laboratory use.
ACKNOWLEDGMENTS This work was supported in part by funds of the “Progetto finalizzato Biotecnologie e Biostrumentazione” of the Italian National Research Council, and by funds of the Italian MURST (40 and 60%). We are indebted to Dr. Alexander A. Chisholm for revising the English form. Patent No. 91A000249.
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