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A Method for Isolation of Small DNA Fragments from Agarose and Polyacrylamide Gels
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NOTES & TIPS A Method for Isolation of Small DNA Fragments from Agarose and Polyacrylamide Gels Jan Kormanec, 1 Dagmar Homerova, Beatrica Sevcikova, and Bronislava Rezuchova Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 842 51 Bratislava, Slovak Republic Received October 12, 2000; published online April 30, 2001
Although a variety of methods exist for isolating DNA fragments from agarose gels, there are problems in most of them with the efficient isolation of small fragments (about 100 bp). There are two wide-spread commercial techniques for isolation of DNA fragments from agarose gels. The technique GeneClean from BIO 101 (La Jolla, CA) employs solubilization with NaI and binding of the DNA to silica-based Glassmilk, and the QIAquick spin kit from Qiagen (Chatsworth, CA) employs a silica-based membrane. During our study we encountered the problem of isolating efficiently small DNA fragments after PCR amplification from agarose gels using both techniques noted above. Although we used a modification of the GeneClean technique for small DNA fragments (2), the yields were too low for efficient blunt-end ligation. In this work we describe a simple method for the efficient isolation of small DNA fragments from both agarose and polyacrylamide gels. We have modified and simplified a method employing the binding of DNA to DEAE 2 paper (3). The protocol was the following: DNA sample after digestion with appropriate restriction enzyme was electrophoresed in agarose gel (usually 0.6 –2%, depending on the length of the isolated fragment) in TAE with 0.5 g/ml ethidium bromide (1). After electrophoresis, the gel was placed on a UV 360 transilluminator, and a slot was cut in front of and behind the DNA fragment of interest. The piece of the DEAE paper (ion-exchange chromatography paper DE81, Whatman, UK) having approximately the same size as the 1
To whom correspondence should be addressed. Fax: (421-7) 54772316. E-mail: [email protected]. 2 Abbreviations used: DEAE, diethylaminoethyl; EDTA, ethylenediamine tetraacetic acid; TAE, Tris-acetate-EDTA; TBE, Tris borate-EDTA. 138
fragment was inserted into the slots, and electrophoresis was continued for another 30 min (the gel was not overlaid by electrophoresis buffer), until the fragment was bound on the DEAE paper (could be checked on UV 360 transilluminator). The DEAE paper with the bound DNA fragment was washed with water, quickly dried on filter paper, and put into the prepared microcentrifuge tube with a small hole in the bottom containing 300 l 1 M NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 8, and standing on the second microcentrifuge tube. The hole in the bottom of the microcentrifuge tube was pierced with a 20 gauge ⫻ 1 21 inch needle, until the tip of the needle was seen inside the tube. The DEAE paper was dispersed by a small tip, and tubes were incubated for 15 min in a 37°C cabinet. The tubes were briefly centrifuged (about 20 s at 10,000g). The eluted fragment passed through the hole into the second microcentrifuge tube, and the DEAE paper remained in the upper tube. For low DNA concentrations (less than 20 g/ml), 10 g (1 l) of glycogen for molecular biology (Boehringer, Mannheim, Germany) was added and DNA was precipitated with 750 l of ethanol for 15 min at ⫺70°C (or 1 h at ⫺20°C). The fragment was centrifuged for 10 min at 10,000g and 2°C, washed with 70% ethanol, dried 2 min in a Speed Vac, and dissolved in TE buffer or water. For very small DNA fragments, DNA sample could be electrophoresed on 5% polyacrylamide gel in TBE (1). After electrophoresis, the gel was stained for 20 min in 0.5 mg/ml ethidium bromide and placed on a UV 360 transilluminator. The region corresponding to the fragment of interest was cut with a scalpel, the piece of the DEAE paper the same size as the DNA band was put on the gel, the piece of the gel was removed, and the same sized Whatman 3MM paper was put on the other side of the gel piece. Such a sandwich was inserted into the wide slot in a 2% agarose gel, and electrophoresis was running in TBE buffer for 30 min at 9 V/cm (not overlaid by electrophoresis buffer). The sandwich was removed, DEAE paper was slid off, and DNA fragment was isolated as above. We have also successfully used this method for isolation of 32P-labeled DNA fragments for S1-nuclease mapping, DNaseI footprinting, and other methods. In this case, the polyacrylamide electrophoresis was also used for isolation. After electrophoresis, the gel (on the Analytical Biochemistry 293, 138 –139 (2001) doi:10.1006/abio.2001.5048 0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
139
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with restriction enzymes, 5⬘-, 3⬘-end labeling with T4 polynucleotide kinase or Klenow fragments of DNA polymerase I, respectively, or random-primed labeling. The glycogen present in the fragment did not interfere with any reaction used. A comparison of the method with the two commercial techniques (GeneClean and QIAquick) is exemplified in Fig. 1. Acknowledgment. This work was supported by Grant 2/7001/20 from the Slovak Academy of Sciences.
REFERENCES 1. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. O., Seidman, J. S., Smith, J. A., and Struhl, K. (1987) Current Protocols in Molecular Biology, Wiley, New York. 2. Smith, L. S., Lewis, T. L., and Matsui, S. M. (1995) Increased yield of small DNA fragments purified by silica binding. BioTechniques 18, 970 –974. 3. Winberg, G., and Hammarskjold, M.-L. (1980) Isolation of DNA from agarose gels using DEAE-paper. Application to restriction site mapping of adenovirus type 16 DNA. Nucleic Acids Res. 8, 253–264.
FIG. 1. Comparison of yields of DNA fragments isolated by DEAE paper and several other methods. One microgram of the ⌽X174 DNA cut with HaeIII was loaded into three lanes of 1.4% agarose gel in TAE with 0.5 g/ml ethidium bromide (1). After a short electrophoresis (DNA samples just entered the gel, bromphenol blue was about 5 mm from the slots), the pieces of the gel containing all DNA fragments from two lanes were removed, and DNA was isolated by commercial kits for isolating DNA fragments from agarose gels. The third lane was used for isolating DNA fragments by DEAE paper. A slot was cut in front of the DNA fragments in gel, a piece of DEAE paper was inserted into the slot, and electrophoresis was continued for 30 min, until all the fragments were bound on the DEAE paper. The fragments were isolated from the DEAE paper as described in the text. The fragments isolated by the particular methods were electrophoresed on the 2% agarose gel. Lanes: 1, 1 g of the ⌽X174 DNA cut with HaeIII; 2, the DNA fragments isolated by QIAquick column (Qiagen); 3, the modification of the GeneClean technique (2); 4, the DNA fragments isolated by DEAE paper.
glass plate) was covered by Saran wrap, labeled by hot ink, and exposed 10 min to autoradiography film. The region corresponding to the fragment of interest was cut with a scalpel, the Saran wrap was removed, and the sandwich with the piece of the DEAE paper and Whatman 3MM paper was prepared as for unlabeled DNA fragment. The rest of the procedure was identical to that for the unlabeled DNA fragment. The method described for isolation of DNA fragments from both agarose and polyacrylamide gels is efficient, simple, and cheap. The yield of the method was about 80 –90% as measured after isolation of 32Plabeled DNA fragments from 100 bp to 4 kb. We have found a similar yield using low-melting-point agarose. The fragments could be used for ligation, digestion
Limitations of Comparative Detection of Proteins via Epitope Tagging Leslie J. Bannon,* M. Sharon Stack,† ,‡ and Kathleen J. Green* ,1 *Department of Pathology and Dermatology, †Department of Obstetrics and Gynecology, and ‡Department of Cell and Molecular Biology, and Robert H. Lurie Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611 Received October 13, 2000; published online May 1, 2001
Epitope tagging affords a powerful means by which to facilitate purification, manipulation, and detection of recombinant proteins. This technique involves the introduction of a short specific sequence at any point within the coding region of the protein of interest, which can later be recognized by an antibody raised against the introduced epitope. Epitope tags are generally small enough so as not to affect the biological activity of the protein and can allow multiple protein species to be detected with the same antibody. An extension of this application is to assess comparative expression levels through detection of the same tag 1 To whom correspondence should be addressed at Department of Pathology W127, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, IL 60611.
Analytical Biochemistry 293, 139 –142 (2001) doi:10.1006/abio.2001.5117 0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
NOTES & TIPS A Method for Isolation of Small DNA Fragments from Agarose and Polyacrylamide Gels Jan Kormanec, 1 Dagmar Homerova, Beatrica Sevcikova, and Bronislava Rezuchova Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 842 51 Bratislava, Slovak Republic Received October 12, 2000; published online April 30, 2001
Although a variety of methods exist for isolating DNA fragments from agarose gels, there are problems in most of them with the efficient isolation of small fragments (about 100 bp). There are two wide-spread commercial techniques for isolation of DNA fragments from agarose gels. The technique GeneClean from BIO 101 (La Jolla, CA) employs solubilization with NaI and binding of the DNA to silica-based Glassmilk, and the QIAquick spin kit from Qiagen (Chatsworth, CA) employs a silica-based membrane. During our study we encountered the problem of isolating efficiently small DNA fragments after PCR amplification from agarose gels using both techniques noted above. Although we used a modification of the GeneClean technique for small DNA fragments (2), the yields were too low for efficient blunt-end ligation. In this work we describe a simple method for the efficient isolation of small DNA fragments from both agarose and polyacrylamide gels. We have modified and simplified a method employing the binding of DNA to DEAE 2 paper (3). The protocol was the following: DNA sample after digestion with appropriate restriction enzyme was electrophoresed in agarose gel (usually 0.6 –2%, depending on the length of the isolated fragment) in TAE with 0.5 g/ml ethidium bromide (1). After electrophoresis, the gel was placed on a UV 360 transilluminator, and a slot was cut in front of and behind the DNA fragment of interest. The piece of the DEAE paper (ion-exchange chromatography paper DE81, Whatman, UK) having approximately the same size as the 1
To whom correspondence should be addressed. Fax: (421-7) 54772316. E-mail: [email protected]. 2 Abbreviations used: DEAE, diethylaminoethyl; EDTA, ethylenediamine tetraacetic acid; TAE, Tris-acetate-EDTA; TBE, Tris borate-EDTA. 138
fragment was inserted into the slots, and electrophoresis was continued for another 30 min (the gel was not overlaid by electrophoresis buffer), until the fragment was bound on the DEAE paper (could be checked on UV 360 transilluminator). The DEAE paper with the bound DNA fragment was washed with water, quickly dried on filter paper, and put into the prepared microcentrifuge tube with a small hole in the bottom containing 300 l 1 M NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 8, and standing on the second microcentrifuge tube. The hole in the bottom of the microcentrifuge tube was pierced with a 20 gauge ⫻ 1 21 inch needle, until the tip of the needle was seen inside the tube. The DEAE paper was dispersed by a small tip, and tubes were incubated for 15 min in a 37°C cabinet. The tubes were briefly centrifuged (about 20 s at 10,000g). The eluted fragment passed through the hole into the second microcentrifuge tube, and the DEAE paper remained in the upper tube. For low DNA concentrations (less than 20 g/ml), 10 g (1 l) of glycogen for molecular biology (Boehringer, Mannheim, Germany) was added and DNA was precipitated with 750 l of ethanol for 15 min at ⫺70°C (or 1 h at ⫺20°C). The fragment was centrifuged for 10 min at 10,000g and 2°C, washed with 70% ethanol, dried 2 min in a Speed Vac, and dissolved in TE buffer or water. For very small DNA fragments, DNA sample could be electrophoresed on 5% polyacrylamide gel in TBE (1). After electrophoresis, the gel was stained for 20 min in 0.5 mg/ml ethidium bromide and placed on a UV 360 transilluminator. The region corresponding to the fragment of interest was cut with a scalpel, the piece of the DEAE paper the same size as the DNA band was put on the gel, the piece of the gel was removed, and the same sized Whatman 3MM paper was put on the other side of the gel piece. Such a sandwich was inserted into the wide slot in a 2% agarose gel, and electrophoresis was running in TBE buffer for 30 min at 9 V/cm (not overlaid by electrophoresis buffer). The sandwich was removed, DEAE paper was slid off, and DNA fragment was isolated as above. We have also successfully used this method for isolation of 32P-labeled DNA fragments for S1-nuclease mapping, DNaseI footprinting, and other methods. In this case, the polyacrylamide electrophoresis was also used for isolation. After electrophoresis, the gel (on the Analytical Biochemistry 293, 138 –139 (2001) doi:10.1006/abio.2001.5048 0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
139
NOTES & TIPS
with restriction enzymes, 5⬘-, 3⬘-end labeling with T4 polynucleotide kinase or Klenow fragments of DNA polymerase I, respectively, or random-primed labeling. The glycogen present in the fragment did not interfere with any reaction used. A comparison of the method with the two commercial techniques (GeneClean and QIAquick) is exemplified in Fig. 1. Acknowledgment. This work was supported by Grant 2/7001/20 from the Slovak Academy of Sciences.
REFERENCES 1. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. O., Seidman, J. S., Smith, J. A., and Struhl, K. (1987) Current Protocols in Molecular Biology, Wiley, New York. 2. Smith, L. S., Lewis, T. L., and Matsui, S. M. (1995) Increased yield of small DNA fragments purified by silica binding. BioTechniques 18, 970 –974. 3. Winberg, G., and Hammarskjold, M.-L. (1980) Isolation of DNA from agarose gels using DEAE-paper. Application to restriction site mapping of adenovirus type 16 DNA. Nucleic Acids Res. 8, 253–264.
FIG. 1. Comparison of yields of DNA fragments isolated by DEAE paper and several other methods. One microgram of the ⌽X174 DNA cut with HaeIII was loaded into three lanes of 1.4% agarose gel in TAE with 0.5 g/ml ethidium bromide (1). After a short electrophoresis (DNA samples just entered the gel, bromphenol blue was about 5 mm from the slots), the pieces of the gel containing all DNA fragments from two lanes were removed, and DNA was isolated by commercial kits for isolating DNA fragments from agarose gels. The third lane was used for isolating DNA fragments by DEAE paper. A slot was cut in front of the DNA fragments in gel, a piece of DEAE paper was inserted into the slot, and electrophoresis was continued for 30 min, until all the fragments were bound on the DEAE paper. The fragments were isolated from the DEAE paper as described in the text. The fragments isolated by the particular methods were electrophoresed on the 2% agarose gel. Lanes: 1, 1 g of the ⌽X174 DNA cut with HaeIII; 2, the DNA fragments isolated by QIAquick column (Qiagen); 3, the modification of the GeneClean technique (2); 4, the DNA fragments isolated by DEAE paper.
glass plate) was covered by Saran wrap, labeled by hot ink, and exposed 10 min to autoradiography film. The region corresponding to the fragment of interest was cut with a scalpel, the Saran wrap was removed, and the sandwich with the piece of the DEAE paper and Whatman 3MM paper was prepared as for unlabeled DNA fragment. The rest of the procedure was identical to that for the unlabeled DNA fragment. The method described for isolation of DNA fragments from both agarose and polyacrylamide gels is efficient, simple, and cheap. The yield of the method was about 80 –90% as measured after isolation of 32Plabeled DNA fragments from 100 bp to 4 kb. We have found a similar yield using low-melting-point agarose. The fragments could be used for ligation, digestion
Limitations of Comparative Detection of Proteins via Epitope Tagging Leslie J. Bannon,* M. Sharon Stack,† ,‡ and Kathleen J. Green* ,1 *Department of Pathology and Dermatology, †Department of Obstetrics and Gynecology, and ‡Department of Cell and Molecular Biology, and Robert H. Lurie Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611 Received October 13, 2000; published online May 1, 2001
Epitope tagging affords a powerful means by which to facilitate purification, manipulation, and detection of recombinant proteins. This technique involves the introduction of a short specific sequence at any point within the coding region of the protein of interest, which can later be recognized by an antibody raised against the introduced epitope. Epitope tags are generally small enough so as not to affect the biological activity of the protein and can allow multiple protein species to be detected with the same antibody. An extension of this application is to assess comparative expression levels through detection of the same tag 1 To whom correspondence should be addressed at Department of Pathology W127, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, IL 60611.
Analytical Biochemistry 293, 139 –142 (2001) doi:10.1006/abio.2001.5117 0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.