- Email: [email protected]
Fast Passive Elution of DNA from Zinc–Imidazole Negatively Stained Polyacrylamide Gels
NOTES & TIPS
Fast Passive Elution of DNA from Zinc±Imidazole Negatively Stained Polyacrylamide Gels Lila R. Castellanos-Serra, Eugenio Hardy,1 and Julio C. Sa´nchez Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana, Cuba Received September 11, 1997
Nucleic acids on polyacrylamide gels are currently detected under UV light of 254 –312 nm after staining with ethidium bromide. They may be recovered from gel slices by passive diffusion at 37°C over a prolonged time, generally overnight (12–16 h) (1). We have recently found that gel-separated nucleic acids can be rapidly (30 min or less) detected by zinc–imidazole negative staining (2). This method affords a sensitivity comparable to that of ethidium bromide, with the significant advantage of avoiding DNA damage and health and environmental hazards due to UV exposure or ethidium bromide handling. Here we present a new method for the enhanced passive elution of negatively stained DNA. This method has the advantage of being fast (about 50 min) and providing a good recovery of DNA (about 90% yield) under normal conditions (room temperature, in the absence of SDS). Methods DNA is detected by a negative staining technique, utilizing zinc–imidazole as described in (2). The new procedure has the following steps: (1) DNA mobilization. The bands of interest are excised, placed into microfuge tubes, incubated (2 3 5 min) in 1 ml, 20 mM Tris–acetate buffer (pH 7.4), 100 mM EDTA to chelate zinc ions, and washed with 1 ml water (2 3 1 min followed by 1 3 10 min). During this step, diffusion of DNA from excised bands to the solution is low (about 6%), as measured by using 32Plabeled DNA in a wide range of molecular weights (from 0.1 to 5 kb). (2) DNA elution. A 32-mm metal sieve disk (Carl Schroeter, Hamburg), cut to fix the internal diameter of a 1-ml polypropylene syringe, was inserted at the bottom. A second 100-mm metal sieve disk was placed over the 32-mm disk and firmly adjusted by pressing them with the aid of the plunger. The bands are transferred to the syringe. The gel is extruded through the sieves by pressing the plunger. Thus, a gel slurry of To whom correspondence should be addressed. Fax: 1537 33 60 08. E-mail: [email protected]. 1
ANALYTICAL BIOCHEMISTRY ARTICLE NO. AB972501
257, 227–228 (1998)
0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
227
32-mm particles is obtained (3, 4). The elution buffer (20 mM Tris–acetate, 0.5 M NH4Ac, 1 mM EDTA, pH 7.4) is added to the syringe (about twice the crushed gel volume, e.g., for a 1- to 5-mg 1-kb DNA band, 100 ml). The solution is flushed through the sieves to remove any gel particle and collected in the microfuge tube containing the slurry. The sample is then incubated for 10 min under permanent vortexing. The tube is centrifuged briefly (approx for 20 s), and the overlaying solution is collected. This elution step is repeated once with elution buffer, followed by an optional wash with water. Alternatively, it is possible to replace centrifugation by filtering the slurry through a polypropylene minicolumn. This minicolumn can be easily prepared by inserting a porous polypropylene disk into a 1-ml polypropylene syringe. The DNA is concentrated and precipitated from the pooled solution by standard procedures. The syringe is thoroughly flushed with 103 Tris– glycine–SDS Laemmli electrophoresis buffer followed by sterile water. In our experience, the syringe can be reused many times. Results and Discussion The negative zinc–imidazole stain immobilizes nucleic acids within the gel matrix, eliminating band diffusion (2). This temporary immobilization is completely reversed by destaining the gel with zinc chelators such as EDTA, after which nucleic acids can be passively eluted. Nucleic acids elute with 90% efficiency from gel bands that have been finely cut to yield 32-mm particles (Fig. 1). Elution efficiency was highly dependent on the gel particle size; yields were only 12% for excised bands that were not further sectioned and 27% for bands that were minced to yield approximately 1-mm3 cubes. Bands that were minced to only 100-mm particles exhibited slower elution kinetics (not shown). For comparison, we evaluated passive elution of DNA from sliced bands after prolonged incubation (18 h at room temperature) in Tris–EDTA buffer. In this case, recovery was slightly lower (76%) than that achieved from the 32-mm gel particles following the present protocol (total elution time about 30 min). Reproducibility. A 650-bp 32P-labeled DNA fragment containing the hu-IFN-a2b gene was electrophoresed in a 5% polyacrylamide gel (1 mg per band, about 20,000 cpm) and detected by zinc–imidazole. Ten bands were independently processed; the average elution efficiency was 90.4% (SD, 1.51; variation coefficient, 1.67%). Time considerations. From gel staining to elution, this protocol can be completed in less that 2 h (approx 30 min corresponds to gel staining, 25 min to zinc chelation and gel crushing, while the elution step takes about 25 min). General applicability of the procedure. The 32P-labeled AvaII restriction digest of bacteriophage l DNA
228
NOTES & TIPS
interfere with DNA polymerase activity, nor does it affect DNA integrity (not shown). Our work is oriented toward the maximum preservation of DNA biological activity. This method combines a sensitive procedure for the negative staining of DNA with a rapid, efficient procedure for DNA elution. REFERENCES 1. Maxam, A. M., and Gilbert, W. (1977). Proc. Natl. Acad. Sci. USA 74, 560 –564. 2. Hardy, E., Pupo, E., Casalvilla, R., Sosa, A. E., Trujillo, L. E., Lo´pez, E., and Castellanos-Serra, L. R. (1996) Electrophoresis 17, 1537–1541. 3. Heukeshoven, J., and Dernik, R. (1991). in Electrophoresis Forum ’91 (Radola, B. J., Ed.), pp. 501–506. 4. Castellanos-Serra, L. R., Ferna´ndez-Patro´n., C., Hardy, E., and Huerta, V. (1996) Electrophoresis 17, 1564 –1572.
FIG. 1. Elution of nucleic acids from zinc–imidazole-stained gels. Identical samples of 32P-labeled DNA fragments (650 bp, 1 mg, about 20,000 cpm) were separated in several lanes of a 5% polyacrylamide– Tris–acetate–EDTA electrophoresis gel. The gel was stained with zinc–imidazole. The DNA bands were excised and processed for DNA mobilization as described under Methods (step 1). One band was not further minced (1). A second band was minced with the aid of a scalpel to yield 1-mm3 cubes (2), The third band was processed as described under Methods (step 2) to yield 32-mm gel particles (3). The bands were incubated in the elution buffer under vortexing (2 3 10 min) (Elution 1 and Elution 2) and then in water (1 3 10 min) (Wash) as described in the text. On-gel radioactivity corresponds to total counts remaining in the gel. Results are the average of three independent experiments.
Determining the Number of Essential Ligand Binding Sites in Enzymes Using the Slopes of the Wang±Srivastava Plots Yunhu Tan Pye Laboratory, CSIRO Land and Water, GPO Box 1666, Canberra, ACT 2601, Australia Received September 11, 1997
was separated in a 5% polyacrylamide gel; 20 bands were resolved (ranging from 0.1 to 5 kb). The lane was sectioned in three regions, corresponding to approximately 0.1– 0.5 kb (low molecular weight), 0.5–1.5 kb (medium molecular weight), and 1.5-5 kb (high molecular weight). DNA recovery was 92% for the low-molecular-weight section and 89% for the medium- and high-molecular-weight sections (data are the average of three independent experiments). High elution yields (.85%) were also found for small (82 bp) DNA separated on a 15% polyacrylamide gel, a 1.3-kb DNA fragment encoding streptokinase, the linearized plasmid cloning vectors pBR322 (4.361 kb), and pUC19 (2.686 kb) separated in a 5% polyacrylamide gel. The integrity of the eluted DNA was confirmed by electrophoretic analysis on agarose or on polyacrylamide gels. The integrity of the fragment ends was verified by standard ligation with T4 DNA ligase (Biolabs) according to the manufacturer’s recommendations, followed by electrophoretic analysis. The biological activity of DNA recovered from gels following this methodology was confirmed by PCR amplification and cloning experiments. This demonstrates that this procedure does not
Wang and Srivastava (1) presented a graphical method for the determination of the number of essential sites for enzymes with multiple binding sites for a ligand (a substrate, an inhibitor, or an activator molecule). This method applies to monomeric enzymes containing multiple unspecific sites and to oligomeric enzymes containing specific sites. Their method was developed for enzyme kinetic models, expressing the initial reaction rate, v, as a ratio of two polynomials in terms of the ligand concentration [L], a class of models common in enzyme kinetics (2, 3). The authors explored the complex mathematical properties of equations of this type. The number of essential ligand binding sites is determined by plotting 1/v[L]p versus [L] for inhibition or [L]p/v versus 1/[L] for activation (p 5 0, 1, 2, . . .). The magnitude of p that yields a horizontal asymptote on those plots corresponds to the number of essential sites. Because it is difficult to determine whether a function yields a horizontal asymptote, this method requires that the concentration of the ligand [L] approach infinity for the case of inhibition or zero for the case of activation to provide a definite assessment of the number of essential ligand binding sites. ANALYTICAL BIOCHEMISTRY 257, 228 –230 (1998) ARTICLE NO. AB972539
0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
Fast Passive Elution of DNA from Zinc±Imidazole Negatively Stained Polyacrylamide Gels Lila R. Castellanos-Serra, Eugenio Hardy,1 and Julio C. Sa´nchez Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana, Cuba Received September 11, 1997
Nucleic acids on polyacrylamide gels are currently detected under UV light of 254 –312 nm after staining with ethidium bromide. They may be recovered from gel slices by passive diffusion at 37°C over a prolonged time, generally overnight (12–16 h) (1). We have recently found that gel-separated nucleic acids can be rapidly (30 min or less) detected by zinc–imidazole negative staining (2). This method affords a sensitivity comparable to that of ethidium bromide, with the significant advantage of avoiding DNA damage and health and environmental hazards due to UV exposure or ethidium bromide handling. Here we present a new method for the enhanced passive elution of negatively stained DNA. This method has the advantage of being fast (about 50 min) and providing a good recovery of DNA (about 90% yield) under normal conditions (room temperature, in the absence of SDS). Methods DNA is detected by a negative staining technique, utilizing zinc–imidazole as described in (2). The new procedure has the following steps: (1) DNA mobilization. The bands of interest are excised, placed into microfuge tubes, incubated (2 3 5 min) in 1 ml, 20 mM Tris–acetate buffer (pH 7.4), 100 mM EDTA to chelate zinc ions, and washed with 1 ml water (2 3 1 min followed by 1 3 10 min). During this step, diffusion of DNA from excised bands to the solution is low (about 6%), as measured by using 32Plabeled DNA in a wide range of molecular weights (from 0.1 to 5 kb). (2) DNA elution. A 32-mm metal sieve disk (Carl Schroeter, Hamburg), cut to fix the internal diameter of a 1-ml polypropylene syringe, was inserted at the bottom. A second 100-mm metal sieve disk was placed over the 32-mm disk and firmly adjusted by pressing them with the aid of the plunger. The bands are transferred to the syringe. The gel is extruded through the sieves by pressing the plunger. Thus, a gel slurry of To whom correspondence should be addressed. Fax: 1537 33 60 08. E-mail: [email protected]. 1
ANALYTICAL BIOCHEMISTRY ARTICLE NO. AB972501
257, 227–228 (1998)
0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
227
32-mm particles is obtained (3, 4). The elution buffer (20 mM Tris–acetate, 0.5 M NH4Ac, 1 mM EDTA, pH 7.4) is added to the syringe (about twice the crushed gel volume, e.g., for a 1- to 5-mg 1-kb DNA band, 100 ml). The solution is flushed through the sieves to remove any gel particle and collected in the microfuge tube containing the slurry. The sample is then incubated for 10 min under permanent vortexing. The tube is centrifuged briefly (approx for 20 s), and the overlaying solution is collected. This elution step is repeated once with elution buffer, followed by an optional wash with water. Alternatively, it is possible to replace centrifugation by filtering the slurry through a polypropylene minicolumn. This minicolumn can be easily prepared by inserting a porous polypropylene disk into a 1-ml polypropylene syringe. The DNA is concentrated and precipitated from the pooled solution by standard procedures. The syringe is thoroughly flushed with 103 Tris– glycine–SDS Laemmli electrophoresis buffer followed by sterile water. In our experience, the syringe can be reused many times. Results and Discussion The negative zinc–imidazole stain immobilizes nucleic acids within the gel matrix, eliminating band diffusion (2). This temporary immobilization is completely reversed by destaining the gel with zinc chelators such as EDTA, after which nucleic acids can be passively eluted. Nucleic acids elute with 90% efficiency from gel bands that have been finely cut to yield 32-mm particles (Fig. 1). Elution efficiency was highly dependent on the gel particle size; yields were only 12% for excised bands that were not further sectioned and 27% for bands that were minced to yield approximately 1-mm3 cubes. Bands that were minced to only 100-mm particles exhibited slower elution kinetics (not shown). For comparison, we evaluated passive elution of DNA from sliced bands after prolonged incubation (18 h at room temperature) in Tris–EDTA buffer. In this case, recovery was slightly lower (76%) than that achieved from the 32-mm gel particles following the present protocol (total elution time about 30 min). Reproducibility. A 650-bp 32P-labeled DNA fragment containing the hu-IFN-a2b gene was electrophoresed in a 5% polyacrylamide gel (1 mg per band, about 20,000 cpm) and detected by zinc–imidazole. Ten bands were independently processed; the average elution efficiency was 90.4% (SD, 1.51; variation coefficient, 1.67%). Time considerations. From gel staining to elution, this protocol can be completed in less that 2 h (approx 30 min corresponds to gel staining, 25 min to zinc chelation and gel crushing, while the elution step takes about 25 min). General applicability of the procedure. The 32P-labeled AvaII restriction digest of bacteriophage l DNA
228
NOTES & TIPS
interfere with DNA polymerase activity, nor does it affect DNA integrity (not shown). Our work is oriented toward the maximum preservation of DNA biological activity. This method combines a sensitive procedure for the negative staining of DNA with a rapid, efficient procedure for DNA elution. REFERENCES 1. Maxam, A. M., and Gilbert, W. (1977). Proc. Natl. Acad. Sci. USA 74, 560 –564. 2. Hardy, E., Pupo, E., Casalvilla, R., Sosa, A. E., Trujillo, L. E., Lo´pez, E., and Castellanos-Serra, L. R. (1996) Electrophoresis 17, 1537–1541. 3. Heukeshoven, J., and Dernik, R. (1991). in Electrophoresis Forum ’91 (Radola, B. J., Ed.), pp. 501–506. 4. Castellanos-Serra, L. R., Ferna´ndez-Patro´n., C., Hardy, E., and Huerta, V. (1996) Electrophoresis 17, 1564 –1572.
FIG. 1. Elution of nucleic acids from zinc–imidazole-stained gels. Identical samples of 32P-labeled DNA fragments (650 bp, 1 mg, about 20,000 cpm) were separated in several lanes of a 5% polyacrylamide– Tris–acetate–EDTA electrophoresis gel. The gel was stained with zinc–imidazole. The DNA bands were excised and processed for DNA mobilization as described under Methods (step 1). One band was not further minced (1). A second band was minced with the aid of a scalpel to yield 1-mm3 cubes (2), The third band was processed as described under Methods (step 2) to yield 32-mm gel particles (3). The bands were incubated in the elution buffer under vortexing (2 3 10 min) (Elution 1 and Elution 2) and then in water (1 3 10 min) (Wash) as described in the text. On-gel radioactivity corresponds to total counts remaining in the gel. Results are the average of three independent experiments.
Determining the Number of Essential Ligand Binding Sites in Enzymes Using the Slopes of the Wang±Srivastava Plots Yunhu Tan Pye Laboratory, CSIRO Land and Water, GPO Box 1666, Canberra, ACT 2601, Australia Received September 11, 1997
was separated in a 5% polyacrylamide gel; 20 bands were resolved (ranging from 0.1 to 5 kb). The lane was sectioned in three regions, corresponding to approximately 0.1– 0.5 kb (low molecular weight), 0.5–1.5 kb (medium molecular weight), and 1.5-5 kb (high molecular weight). DNA recovery was 92% for the low-molecular-weight section and 89% for the medium- and high-molecular-weight sections (data are the average of three independent experiments). High elution yields (.85%) were also found for small (82 bp) DNA separated on a 15% polyacrylamide gel, a 1.3-kb DNA fragment encoding streptokinase, the linearized plasmid cloning vectors pBR322 (4.361 kb), and pUC19 (2.686 kb) separated in a 5% polyacrylamide gel. The integrity of the eluted DNA was confirmed by electrophoretic analysis on agarose or on polyacrylamide gels. The integrity of the fragment ends was verified by standard ligation with T4 DNA ligase (Biolabs) according to the manufacturer’s recommendations, followed by electrophoretic analysis. The biological activity of DNA recovered from gels following this methodology was confirmed by PCR amplification and cloning experiments. This demonstrates that this procedure does not
Wang and Srivastava (1) presented a graphical method for the determination of the number of essential sites for enzymes with multiple binding sites for a ligand (a substrate, an inhibitor, or an activator molecule). This method applies to monomeric enzymes containing multiple unspecific sites and to oligomeric enzymes containing specific sites. Their method was developed for enzyme kinetic models, expressing the initial reaction rate, v, as a ratio of two polynomials in terms of the ligand concentration [L], a class of models common in enzyme kinetics (2, 3). The authors explored the complex mathematical properties of equations of this type. The number of essential ligand binding sites is determined by plotting 1/v[L]p versus [L] for inhibition or [L]p/v versus 1/[L] for activation (p 5 0, 1, 2, . . .). The magnitude of p that yields a horizontal asymptote on those plots corresponds to the number of essential sites. Because it is difficult to determine whether a function yields a horizontal asymptote, this method requires that the concentration of the ligand [L] approach infinity for the case of inhibition or zero for the case of activation to provide a definite assessment of the number of essential ligand binding sites. ANALYTICAL BIOCHEMISTRY 257, 228 –230 (1998) ARTICLE NO. AB972539
0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.