This article reprinted from: Dooley, M. M. 2008. Restriction endonuclease digestion of a plasmid. Pages 389-392, in Tested Studies for Laboratory Teaching, Volume 29 (K.L. Clase, Editor). Proceedings of the 29th Workshop/Conference of the Association for Biology Laboratory Education (ABLE), 433 pages. Compilation copyright 2008 by the Association for Biology Laboratory Education (ABLE) ISBN 1-890444-11-1 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the copyright owner. Use solely at one s own institution with no intent for profit is excluded from the preceding copyright restriction, unless otherwise noted on the copyright notice of the individual chapter in this volume. Proper credit to this publication must be included in your laboratory outline for each use; a sample citation is given above. Upon obtaining permission or with the sole use at one s own institution exclusion, ABLE strongly encourages individuals to use the exercises in this proceedings volume in their teaching program. Although the laboratory exercises in this proceedings volume have been tested and due consideration has been given to safety, individuals performing these exercises must assume all responsibilities for risk. The Association for Biology Laboratory Education (ABLE) disclaims any liability with regards to safety in connection with the use of the exercises in this volume. The focus of ABLE is to improve the undergraduate biology laboratory experience by promoting the development and dissemination of interesting, innovative, and reliable laboratory exercises. Visit ABLE on the Web at: http://www.ableweb.org
Restriction Endonuclease Digestion of a Plasmid Margaret M. Dooley Biology Department College of Staten Island The City University of New York 2800 Victory Boulevard, Staten Island, NY 10314 dooley@mail.csi.cuny.edu Abstract: Recombinant DNA technology is widely utilized and therefore, undergraduate laboratory courses incorporate these techniques. This reliable laboratory exercise introduces the student to plasmid vectors, restriction enzymes, and agarose gel electrophoresis. Restriction digestion of pbr325 with PstI and HindIII, in single and double digests, was performed and analyzed on an agarose gel. Students constructed a standard curve based on the migration of fragments from a HindIII digest of bacteriophage λ DNA and used that curve to estimate the sizes of DNA fragments in the pbr325 digests. In this exercise, measurements on the largest λ fragment were included to illustrate the resolution limits of agarose gels and the proper use of standard curves. Introduction The plasmid cloning vector, pbr325, carries genes coding resistance to the antibiotics tetracycline, ampicillin and chloramphenicol. Unique restriction sites are located in each antibiotic resistance gene, for example, PstI in ampicillin, BamHI in tetracycline and EcoRI in chloramphenicol (Bolivar, 1978; Prentki et al., 1981) and at other positions around the plasmid, e.g., HindIII which is located in the promoter for the tetracycline resistance gene (Rodriguez et al., 1979). The location of unique restriction sites at various positions around the plasmid permits the formation of easily visualized and well separated restriction fragments. Thus, this vector is well suited for use in instructional restriction analysis protocols. Using this vector also introduces the student to the structure and utilization of the widely utilized plasmid cloning vector pbr322 and its derivatives. Students digest pbr325 with HindIII and PstI, perform agarose gel electrophoresis, and estimate the fragment sizes from a standard curve generated from a λ HindIII digest. In this exercise, this plot includes the 23,130 base pair (bp) λ fragment to illustrate the limited resolution range of agarose gels and the necessity of having points on the standard curve bracket the experimental measurements. Materials and Methods Materials and Solutions Restriction enzymes, restriction buffers, and λ DNA were from New England BioLabs; pbr325 was from Sigma-Aldrich. Plasmid DNA was resuspended in sterile TE buffer to 0.1 µg/µl, and λ DNA to 0.4 µg/µl. Electrophoresis reagents (agarose, loading dye, Tris-Borate-EDTA (TBE) buffer, ethidium bromide solution) and Tris-EDTA buffer can be purchased from Carolina Biological Supply Company or prepared according to Sambrook et al. (1989). Tris-EDTA (TE) is
390 ABLE 2007 Proceedings Vol. 29 10mM Tris-HCl (ph 8.0), 1mM EDTA (ph 8.0); 5X TBE stock solution is 54 g Tris base, 27.5 g boric acid, 20 ml 0.5 M EDTA (ph8.0); loading dye is 0.25% bromophenol blue, 0,25% xylene cyanol FF, 40% (w/v) sucrose in water (Sambrook et al., 1989). Safety Students were carefully supervised when they started the electrophoresis to prevent any safety hazards. Staining was with 1 µg/ml ethidium bromide which is a mutagen and suspect carcinogen. Therefore, to minimize risk, students were not allowed to handle the staining solution, aerosols were avoided, and a 5 mg/ml stock solution was purchased, not a powder. Ethidium bromide solution was chemically inactivated before disposal (Quillardet and Hofnung, 1988) or collected on commercially available filters which were discarded with hazardous waste. To protect the retina and skin from shorter wavelength ultraviolet light, an ultraviolet absorbing shield was always used when viewing gels. Experimental Procedure Students added all reagents as listed in Table 1 to digest plasmid (0.5 µg) with HindIII and PstI in both single and double digests. A HindIII digest of λ DNA (2 µg) was also performed to provide molecular weight standards on the agarose gel. After incubation for 1 hour at 37 C, samples were heated to 60 C for 3 minutes to melt the λ cohesive ends before loading on a 0.8% agarose gel and performing electrophoresis at 80-100 volts for approximately 1 hour until the dye front was 1-2 cm from the end of the gel. If the experiment could not be completed in one laboratory session, samples were frozen after the restriction digestion and the 60 C incubation and electrophoresis was performed during a second session. The technician stained the gels in 1 µg/ml ethidium bromide for at least 1 hour and rinsed with water before viewing on an ultraviolet emitting transilluminator. The gels were photographed with Polaroid 667 film. The fragment migration distances were measured on an 8 X 11 inch print of the digitized photograph or alternatively, an expanded copy, or the Polaroid print itself. Table 1. Restriction reaction mixes Tube # Water Buffer DNA Enzyme 1 10 µl 4 µl 5X HindIII buffer 5 µl λ 1 µl HindIII 2 10 µl 4 µl 5X PstI buffer 5 µl plasmid 1 µl PstI 3 9 µl 4 µl 5X HindIII buffer 5 µl plasmid 1 µl PstI AND 1 µl HindIII 4 10 µl 4 µl 5X HindIII buffer 5 µl plasmid 1 µl HindIII 5 11 µl 4 µl 5X HindIII buffer 5 µl plasmid --- Results and Discussion The results of an ideal gel are shown in Figure 1A. Comparison of either single digest with the λ ladder confirms the size of pbr325 which has 5,995 base pairs (bp). The double digest
Poster Session 391 produces fragments that are approximately 2,400 and 3,600 bp as expected. Clearly, HindIII and PstI each cleave pbr325 only once and at different sites on the plasmid. Most undigested plasmid migrates closer to the wells than the linear pbr325 in the single digest lanes, confirming that both restriction enzymes cleaved pbr325 and emphasizing the need to compare like conformations in size determinations. 1A 1B Figure 1A. Agarose gel electrophoresis of restriction digests. Lane 1, λ DNA digested with HindIII (the size of each fragment is indicated in base pairs); lane 2, pbr325 digested with PstI; lane 3, pbr325 digested with PstI and HindIII; lane 4, pbr325 digested with HindIII; and lane 5, undigested pbr325. 1B. Standard curve for fragment size determination. The distance each λ HindIII fragment migrated was measured and plotted (arithmetic X-axis) verses the size in base pairs (logarithmic Y-axis).
392 ABLE 2007 Proceedings Vol. 29 Figure 1B shows the standard curve semilog plot that was used to determine the fragment sizes. A 0.8% gel cannot resolve fragments in the 23,130 bp range and therefore, this λ fragment is not on the linear portion of the plot which extends from 2,027 to at most 9,416 bp. This can be used to illustrate the importance of bracketing the unknown points with the standard curve as a linear relationship might not continue beyond the measured range. In the case of the λ HindIII ladder, if the 2,027-9,416 bp linear portion of the curve were incorrectly extrapolated beyond the highest point, the apparent size of the 23,130 bp fragment would be 12,000 bp, nearly a two fold error. Clearly, it is inappropriate to extrapolate a standard curve beyond the measured range. The 2,027-9,416 bp linear portion of the standard curve also reflects the fragment sizes that can be properly resolved; the steeper segment shows the size where the gel can no longer suitably separate fragments. The lack of resolution of larger DNA s can be emphasized by asking students to predict how far 23,000 and 22,700 bp fragments would migrate (both 62 mm) compared to the easily resolved 2,322 and 2,027 bp fragments (110 and 115 mm). Assessment was from grading of student lab reports and a quiz. In this exercise, students gained knowledge of the pbr325 cloning vehicle, the restriction digestion procedure, the agarose gel electrophoresis procedure, the interpretation of agarose gel patterns, and certain dilution procedures. The resolution limits of agarose gels and the correct use of standard curves were emphasized by considering the data on the largest λ fragment. In addition, students acquired the information and practice required to properly use micropipettors, microcentrifuges, and electrophoresis equipment. Literature Cited Bolivar, F. 1978. Construction and Characterization of new cloning vehicles. Gene 4:121-136. Prenrki, P., F. Karch, S. Iida and J. Meyer. 1981. The plasmid cloning vector pbr325 contains a 482 base pair long inverted duplication. Gene 14:289-299. Quillardet, P. and M. Hofnung. 1988. Ethidium bromide and safety-readers suggest alternative solutions. (Letter to editor) Trends in Genetics 4: 89. Rodriguez, R. L., R. W. West, H. L. Heyneker, F. Bolivar, H. W. Boyer. 1979. Characterizing wildtype and mutant promoters of the tetracycline resistance gene in pbr313. Nucleic Acids Research 6:3267-87. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning A Laboratory Manual. Book 3. Second Edition. Cold Spring Harbor Laboratory Press, 1659 pages. About the Author Margaret M. Dooley earned her B.A. from Cornell University and her Ph.D. from Syracuse University. She received postdoctoral training at the University of California, Davis and Rutgers University before joining the faculty of the College of Staten Island where she teaches genetics, microbiology and general biology courses. 2007 Margaret M. Dooley
Restriction Endonuclease Digestion of a Plasmid Margaret M. Dooley, Biology Department, College of Staten Island/The City University of New York, Staten Island, NY, 10314 dooley@mail.csi.cuny.edu Abstract Recombinant DNA technology is widely utilized and therefore, undergraduate laboratory courses incorporate these techniques. This reliable laboratory exercise introduces the student to plasmid vectors, restriction enzymes, and agarose gel electrophoresis. Restriction digestion of pbr325 with PstI and HindIII, in single and double digests, was performed and analyzed on an agarose gel. Students constructed a standard curve based on the migration of fragments from a HindIII digest of DNA and used that curve to estimate the sizes of DNA s in the pbr325 digests. Results from the gel were as expected: PstI and HindIII single digests produced one band at approximately 6000 bp confirming the size of pbr325 and the double digest produced two fragments approximately 2400 and 3600 bp. Clearly, these restriction endonucleases that cleave pbr325 only once are located at different positions on the plasmid. Questions to promote further thought and reinforce mathematics skills were included at the end of the exercise. In particular, the necessity of having points on the standard curve bracket the unknown data is emphasized by considering the apparent molecular weight of the 23,130 bp fragment. Fragments of this size cannot be properly resolved on this type of gel. If this band were of unknown size, extrapolation of the linear portion of the standard curve would estimate its size to be approximately 12,000 bp. Clearly, it is inappropriate to extrapolate a standard curve beyond the measured range. The linear portion of the standard curve also reflects the fragment sizes that can be properly resolved; the curved segment shows the size where the gel can no longer suitably separate fragments. Materials and Methods Materials and Solutions Restriction enzymes, restriction buffers, and DNA were from New England BioLabs; pbr325 was from Sigma-Aldrich. Plasmid DNA was resuspended in sterile TE buffer to 100 g/ml (0.1 g/ l) and DNA to 400 g/ml (0.4 g/ l). Electrophoresis reagents (agarose, loading dye, Tris-Borate-EDTA (TBE) buffer, ethidium bromide solution) and Tris-EDTA buffer can be purchased from Carolina Biological Supply Company or prepared according to Sambrook et al., 1989. Safety Students were carefully supervised when they started the electrophoresis to prevent any safety hazards. Staining was with 1 g/ml ethidium bromide which is a mutagen and suspect carcinogen. Therefore, to minimize risk, students were not allowed to handle the staining solution, aerosols were avoided and a 5 mg/ml stock solution was purchased, not a powder. Ethidium bromide solution was chemically inactivated before disposal (Quillardet, 1988) or collected on commercially available filters which were discarded with hazardous waste. To protect the retina and skin from shorter wavelength UV light, a UV absorbing shield was always used when viewing gels. Experimental Procedure Students added all reagents as listed in Table I to digest plasmid (0.5 g) with HindIII and PstI in both single and double digests. A HindIII digest of DNA (2 g) was also performed to provide molecular weight standards on the agarose gel. After incubation for 1 hour at 37 C, samples were heated to 60 C for 3 minutes to melt the cohesive ends before loading on a 0.8% agarose gel and performing electrophoresis at 80-100V approximately 1 hour until the dye front was 1-2 cm from the end of the gel. If the experiment could not be completed in one laboratory session, samples were frozen after the restriction digestion and the 60 C incubation and electrophoresis was performed during a second session. The technician stained the gels in 1 g/ml ethidium bromide for at least 1 hour and rinsed with water before photographing with Polaroid 667 film. The fragment migration distance was measured on a 8 X 11 print of the digitized photograph. Alternatively, an expanded copy or the Polaroid print could be measured. Assessment was from grading of student lab reports and a quiz that included questions about restriction digestion, use of plasmids in cloning experiments, agarose gel electrophoresis, and dilution of buffers. Students gained knowledge of (i) the specificity of restriction enzymes, (ii) the composition of restriction digestion reaction mixes, (iii) the agarose gel electrophoresis procedure, (iv) the interpretation of agarose gel patterns, (v) certain dilution procedures, and (vi) laboratory skills such as using micropipettors, microfuges, and electrophoresis equipment. Details of the student protocol and results will be presented. Introduction The plasmid cloning vector, pbr325, carries genes coding resistance to the antibiotics tetracycline, ampicillin and chloramphenicol. Unique restriction sites are located in each antibiotic resistance gene, for example, PstI in ampicillin, BamHI in tetracycline and EcoRI in chloramphenicol (Bolivar,1978; Prentki, 1981) and at other positions around the plasmid, e.g., HindIII which is located in the promoter for the tetracycline resistance gene (Rodriguez et al., 1979). The location of unique restriction sites at various positions around the plasmid permits the formation of easily visualized and well separated restriction fragments. Thus, this vector is well suited for use in instructional restriction analysis protocols. Using this vector also introduces the student to the structure and utilization of the widely utilized plasmid cloning vector pbr322 and its derivatives. Students digest pbr325 with HindIII and PstI, perform agarose gel electrophoresis, and estimate the fragment sizes from a standard curve generated from a HindIII digest. Results and Discussion The results of an ideal gel are shown in Figure 1. Comparison of either single digest with the ladder confirms the size of pbr325 which has 5995 bp. The double digest produces fragments that are approximately 2400 and 3600 bp as expected. Most undigested plasmid migrates closer to the wells than the linear pbr325 in the single digest lanes, confirming that both restriction enzymes cleaved pbr325 and emphasizing the need to compare like conformations in size determinations. Figure 2 shows the standard curve semilog plot that was used to determine the fragment sizes. A 0.8% gel cannot resolve fragments in the 23130 bp range and therefore, this fragment is not on the linear portion of the plot. This can be used to illustrate the importance of bracketing the unknown points with the standard curve as a linear relationship might not continue beyond the measured range. In the case of the HindIII ladder, if the linear portion of the curve were incorrectly extrapolated beyond the 9416 bp point, the apparent size of the 23130 bp fragment would be 12,000 bp, nearly a two fold error. Also, the lack of resolution of larger DNA s can be emphasized by asking students to predict how far 23000 and 22700 bp fragments would migrate (both 62 mm) compared to the easily resolved 2322 and 2027 bp fragments (110 and 115 mm). Thus, this exercise can be used to introduce standard curves, agarose gel electrophoresis, and restriction digestion. References Bolivar, F. 1978. Construction and Characterization of new cloning vehicles Gene 4:121-136. Prenrki, P., F. Karch, S. Iida and J. Meyer. 1981. The plasmid cloning vector pbr325 contains a 482 base-pair-long inverted duplication. Gene 14:289-299. Quillardet, P. and Hofnung. 1988. Ethidium bromide and safety-readers suggest alternative solutions.(letter to editor) Trends in Genetics 4: 89. Rodriguez, R. L., West, R. W., Heyneker, H. L., Bolivar F, Boyer, H. W. 1979. Characterizing wild-type and mutant promoters of the tetracycline resistance gene in pbr313. Nucleic Acids Res. 6:3267-87. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning A Laboratory Manual Second Edition. Cold Spring Harbor Laboratory Press