[2]
PREPARING
AND USING AGAROSE MICROBEADS
13
[2] P r e p a r i n g a n d U s i n g Agarose Microbeads B y MICHAEL KOOB and WACLAW SZYBALSKI
Introduction Protocols for preparing and manipulating large DNA molecules without breakage are required for the routine analysis of genomes by pulsed-field gel electrophoresis (PFGE). To protect genomic DNA from shear, cells are typically embedded in agarose blocks before lysis and deproteinization. 1A less commonly used alternative means of preparing stable genomic DNA was devised by Cook to study the chromatin structure of human chromosomes. 2,3 This process starts with whole cells being embedded in agarose microbeads,4'5 which are essentially minute agarose blocks, and is followed by cell lysis and release of DNA, which remains trapped in the microbeads. Several adaptations of Cook's original microbead procedure to pulsedfield gel applications have been described. 6-8 Because of their small size, agarose microbeads potentially offer several advantages over agarose blocks, both in ease of handling and in the speed with which the DNA can be prepared and enzymatically manipulated. Despite this, however, microbeads have not been widely considered to be satisfactory replacements for agarose plugs. This is due for the most part to difficulties many researchers have reported when working with microbead-embedded DNA. Once washed, agarose microbeads tend to stick to plasticware, pipette tips, and to each other, thus complicating manipulations. When loaded in wells and electrophoresed, they often produce bands that are diffuse or streaked. Finally, when cells are embedded at too high a density, trapping and background smearing tend to occur more frequently as compared with DNA embedded in agarose plugs. In the course of our work to develop new genomic cleavage techniques, we have developed a comprehensive, simple set of procedures for prepar1 D. C. Schwartz and C. R. Cantor, Cell (Cambridge, Mass.) 37, 67 (1984). 2 p. R. Cook, EMBO J. 3, 1837 (1984). 3 D. A. Jackson and P. R. Cook, EMBO J. 4, 913 (1985). 4 K. Nilsson, W. Scheirer, O. W. Merten, L. Ostberg, E. Liehl, H. W. D. Katinger, and K. Mosbach, Nature (London) 302, 629 (1983). 5 K. Nilsson, W. Scheirer, H. W. D. Katinger, and K. Mosbach, this series, Vol. 121, p. 352. 6 G. F. Carle and M. V. Olson, this series, Vol. 155, p. 468. 7 M. McClelland, this series, Vol. 155, p. 22. 8 p. j. Piggot and C. A. M. Curtis, J. Bacteriol. 169, 1260 (1987).
METHODS IN ENZYMOLOGY, VOL. 216
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
14
ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A
[2]
ing and enzymatically manipulating genomic DNA in agarose microbeads that reproducibly overcome the major problems typically associated with these techniques. 9 Incubation steps for lysis, deproteinization, and enzymatic modification are short, the microbeads are easy to handle, and the quality of the resulting pulsed-field gels is usually superior to those typically obtained with agarose plugs. Although the details in the protocol given are for the preparation of Escherichia coli and Saccharomyces cerevisiae genomic DNA, these procedures can be readily applied to most types of cells. Adaptation of the appropriate steps is discussed. Similarly, the protocol for the enzymatic digestion of the microbead-embedded DNA can be easily adapted to almost any other enzymatic modification (e.g., methylationg).
Preparing and Using Agarose Microbeads Solutions Escherichia coil lysis buffer (ELB): Prepare a buffer containing 1 M
NaCI, 0.1 M ethylenediaminetetraacetic acid (EDTA; pH 8.0/25°), 10 mM Tris-HCl (pH 8.0/25°), and 1% (w/v) sodium N-lauroylsarcosine with sterile stock solutions and store at room temperature. Add 1 mg lysozyme (Sigma, St. Louis, MO) and 2 ~1 RNase (10 mg/ml DNasefree stock, stored at 4 °) per milliliter of lysis buffer just before use Yeast spheroplast buffer (YSB): Prepare a solution of three parts sterile SCE (shown below) and two parts sterile 0.5 M EDTA (pH 8.0/25 °) and store at room temperature. Resuspend microbead-embedded yeast in 4 ml of this buffer and just before incubation at 37° add 1 mg lyticase (about 1000 units; Sigma) and 200 ~1 2-mercaptoethanol (about 14.4 M; Sigma) to this suspension. (The Corex tube should be covered with Parafilm during incubation because of the 2-mercaptoethanol) ES: Add 1% (w/v) sodium N-lauroylsarcosine to sterile 0.5 M EDTA (pH 8/25 °) and store at room temperature ESP: Add 1 mg proteinase K (Sigma) per milliliter ES and incubate at 37 ° for 0.5 hr to eliminate DNases. Prepare this solution just before use SCE: Prepare a buffer consisting of 1.0 M sorbitol, 0.1 M sodium citrate, and 60 mM EDTA, adjust to pH 7.0/25 ° with HC1, sterilize, and store at room temperature TE: I0 mM Tris-HCl, 1 mM EDTA, pH 8.0/25 ° 9 M. Koob and W. Szybalski, Science 250, 271 (1990).
[2]
PREPARING AND USING AGAROSE MICROBEADS
15
STE: Dilute sterile 5 M NaC1 stock to 1 M TE PMSF/TE: Prepare a 100 mM phenylmethylsulfonyl fluoride (PMSF) stock by dissolving 26 mg PMSF in 1.5 ml ethanol and store at - 20°. Dilute to 1 mM with TE just before use. (PMSF is toxic and should be handled with care) TEX, 0.1EX, and 0.5EX: Add 0.01% (v/v) Triton X-100 (Sigma) to sterile TE, 0.1 M EDTA (pH 8.0/25°), and 0.5 M EDTA (pH 8.0/25°), respectively, and store at room temperature
Cell Preparation Cells are grown and prepared for embedding in microbeads in essentially the same manner as for agarose plugs. In every case they are washed once in a buffer in which they are stable and then concentrated to twice the final desired concentration in the same buffer. Escherichia coli. Inoculate 50 ml LB (5 g yeast extract, 10 g tryptone, 10 g NaCI, distilled water to 1 liter) with 1 ml overnight culture and shake at 37° until the OD550 is 0.2-0.3. Add chloramphenicol to a final concentration of 180 ~g/ml to stop chromosomal replication and aid in cell lysis. Again incubate at 37° until growth has stopped (0.5-1 hr). Chill on ice, determine the final OD550, and calculate the number of cells per milliliter by assuming that there are 1 × 108 cells/ml at an OD550of 0.24. Concentrate 1-2 × 10 9 cells by centrifugation, wash once with 5 ml STE, and resuspend in STE to a final volume of 2 ml. (NotI-digested DNA from microbeads made with 1 × 109 cells will give fine, light DNA bands on PFGE, and that from microbeads made with 2 × 10 9 cells will give intense, heavy bands.) Saccharomyces cerevisiae. Grow cells by inoculating 10 ml YPD (10 g yeast extract, 20 g peptone, 20 g glucose, distilled water to 1 liter) with a fresh culture and shake overnight at 30°. Wash the cells once with 5 ml SCE and resuspend in SCE to a final volume of 2 ml.
Embedding Cells in Microbeads 1. Add 5 ml of paraffin oil (or light mineral oil) to a sterile 25-ml Pyrex flask and warm to 42 °. Prepare a 1% (w/v) solution of low-melting-point (LMP) agarose (e.g., InCert agarose; FMC, Rockland, ME) in glass-distilled sterile H20 and cool to 50 ° [see (a) in the following section]. 2. Warm the 2 ml of washed cells to room temperature and add them to 2 ml 50° agarose. Swirl briefly to mix, and pour into the warm paraffin oil. 3. Vortex the oil/agarose vigorously [see (b) in the following section] until a fine, milky emulsion has formed (30-60 sec). Immediately swirl the
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ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A
[2]
flask in an ice/salt water bath (1 min) to cool the oil quickly and solidify the agarose beads. 4. To remove the oil, add 5 ml 0.1EX to the oil/microbeads emulsion, vigorously swirl the flask, and pour the suspension into a 15-ml Corex centrifuge tube. Pellet the microbeads by centrifugation (5 min at 5000 rpm in a Sorvall (Norwalk, CT) SS-34 rotor, 4°). Carefully pour the oil and buffer off the pellet.
Notes on Embedding Cells a. When enzymatic reactions will be performed that require incubation above 55 °, a highly purified normal-melting-point agarose (e.g., GTG agarose; FMC) may be used in place of LMP agarose in the above procedure. However, when this is done, the temperature of the cells, oil, and agarose should be increased to 37, 50, and 50°, respectively. This keeps the agarose from setting before the microbeads have formed. In addition, deproteinization (below) can be carried out at 65 °. b. Vigorous vortexing and rapid cooling (step 3) are the most critical steps in making uniform preparations of small microbeads. The best results are obtained when the flask is held nearly horizontal with its base pressed firmly to the vibrating nub, causing the liquid to froth violently. To entrap larger cells, such as those from human tissue culture, less vigorous mixing should be used to produce slightly larger microbeads. c. Attempting to embed cells at unusually high concentrations will result in large numbers of cells trapped on the microbead surface and free in solution. This in turn will lead to severe clumping and, following cell lysis, an unusually viscous solution. Discard preparations that show these symptoms and repeat the embedding process at a lower cell concentration.
Cell Lysis and Deproteinization 1. To remove the cell walls from the embedded cells, resuspend the microbead pellet in 4 ml of the appropriate buffer (ELB or Y S B - - n o t e that the lyticase and 2-mercaptoethanol are added only at this point) and incubate at 37° for 0.5 hr (E. coli) or 1 hr (yeast). Pellet the microbeads and pour off the buffer [see (a)-(c) in the following section]. 2. Resuspend the pellet in 4 ml ESP, incubate at 52° for 1 hr, and then remove the ESP [see (c) in the following section]. 3. Wash the deproteinized pellet once with 8 ml TE, twice with 4 ml PMSF/TE, once or twice with 8-10 ml TE, and then once with 2 ml 0.5EX. Take care to remove all of the EX from above the pellet after the last wash. 4. To check the quality of the preparation, draw 200/xl of the micro-
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PREPARING AND USING AGAROSE MICROBEADS
17
beads from the pellet into an uncut Gilson P-200 disposable pipette tip (Rainin, Emeryville, CA) and then expell them back to the pellet. They should pipette smoothly and have a consistency similar to that of a heavy oil. If they do not, they either (1) have clumps of microbeads, or (2) are too dry. To eliminate small clumps of microbeads, repeatedly draw them into and forcefully expell them from the pipette tip until they flow smoothly [see (d) in the following section]. In the case of an overly dry pellet, add enough 0.5EX back to the pellet to allow the microbeads to be pipetted. 5. Cover the Corex tube with Parafilm and store the microbeads at 4° [see (e) in the following section]. Notes on Preparing Genomic DNA a. The enzymes and buffers used in step 1 should be appropriately modified for removing the cell wall from other types of cells. Microbeadembedded cells without cell walls are treated directly with ESP (i.e., a separate lysis step is not necessary). b. For incubation during the lysis and deproteinization steps, the Corex tube containing the resuspended microbeads is placed in the appropriate water bath. Shaking is not necessary. c. To change a buffer, pellet the microbeads by centrifugation (5 min at 5000 rpm in a Sorvall SS-34 rotor, 4°) and pour the buffer carefully off the pellet. Add the new buffer and completely resuspend the pelleted microbeads by vigorous vortexing. Clumps of microbeads that do not separate during vortexing should be broken by pipetting up and down through a disposable transfer pipette. d. In addition to making the microbeads easy to pipette, disrupting microbead clumps allows for more uniform and efficient enzymatic treatment of the DNA in the microbeads and minimizes trapping during pulsedfield gel electrophoresis. The Triton X-100, which does not inhibit most enzymatic reactions at low concentrations, prevents the microbeads from sticking to themselves and to the plastic and glassware with which they came in contact. Incorporation of small amounts of this nonionic detergent in many of the solutions throughout this procedure (as indicated) is critical. e. Although genomic DNA prepared in this way can be stored at 4° and used successfully for over a year, the best results are obtained from microbeads less than a few months old. Enzymatic Digestion of Microbead-Embedded DNA 1. Pipette 20-30/zl of microbeads into a clear or light-colored Eppendoff microfuge tube [see (a) in the following section]. 2. Wash the beads once with 200/zl TEX and then twice with 200/~1
18
ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A
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of the supplier's recommended digestion buffer without bovine serum albumin (BSA) [see (b) in the following section]. 3. Add 0.1 vol BSA (2-3/zl of sterile 1 mg/ml stock solution) and 0.5-1 /xl restriction enzyme to the buffer-equilibrated pellet and mix thoroughly with the end of the pipette tip. Place the microfuge tube in the appropriate water bath and incubate for 1 hr (shaking is not necessary). 4. If the microbeads will be loaded on a gel immediately after the digestion, wash the pellet with 200/zl TEX. For samples that are to be stored for longer than a few hours before loading, add an equal volume of ES to the digest, incubate at 52° for 15 min to inactivate all nucleases completely, and store the sample at 4 °.
Notes on Enzymatic Manipulation of DNA in Microbeads a. Using Gilson P-200 disposable pipette tips, from each of which approximately 5 mm of the end has been removed (use a razor blade), transfer small amounts of microbead suspensions. The larger bore of such pipette tips allows the slow-flowing microbead suspension to be more easily and accurately pipetted. b. To wash the microbead pellet, forcefully pipette TEX or buffer into the tube (the force of the buffer injected into the tube is sufficient to resuspend the microbeads completely), centrifuge for 1 min in a microfuge at maximum speed, and remove the buffer down to the microbead pellet by pipetting with an uncut tip. Although microbeads made from yeast have a slightly milky appearance, those made from E. coli are clear and difficult to see. To distinguish the clear microbead pellet from the buffer, hold the Eppendorf tube up to a light to make the pellet/buffer boundary visible by the difference in diffraction. The buffer remaining in the microbead pellet after the washing process is sufficient for the enzymatic reaction. c. The above protocol can be used for sequential digestions and for enzymatic manipulations other than digesting. In each case, wash the pellet once with TEX (to remove the previous buffer and to coat the microbeads with Triton X-100) and then twice with the appropriate buffer. Add the BSA, enzyme, and other necessary reagents directly to the bufferequilibrated pellet.
Pulsed-Field Gel Electrophoresis (PFGE) 1. Prepare (a) an agarose solution suitable for PFGE in a 250-ml Pyrex Erlenmeyer flask and (b) a small amount of a 0.5% solution of the same agarose and place them in a 50° water bath until needed [see (a) in the following section]. 2. Add 10/xl TE to the TEX-washed microbead pellet and mix with
[2]
19
P R E P A R I N G A N D U S I N G AGAROSE MICROBEADS
COMB
~ 0 ["
~
~
0 ~
o ~t~
.o,~ t:~
A~ro~ Immersion ~ Level
. . . . . . .
- . . . . .
,~?~7~7
N F1G. 1. L o a d i n g m i c r o b e a d s on t h e t e e t h o f a c o m b (top v i e w ) .
the pipette tip. Load the TE/microbead suspension directly on a tooth of the (horizontal) comb using a cut pipette tip [see (a) in the preceding section]. Form a uniform, fiat droplet completely covering the portion of the tooth face that will be immersed in agarose (see Fig. 1). Repeat for each sample. 3. Once all samples have been loaded on the teeth, remove the TE by touching the lower edge of the microbead/TE droplet with the edge of a small piece of absorbent paper towel cut to the width of the tooth. Hold the towel against the edge of the tooth until all of the TE has been absorbed from the sample into the towel (about 15 sec) and the texture of the microbeads is visible. If necessary, stray microbeads may be pushed into place with the paper towel at this time. Add 5-10/~1 of the 0.5% agarose solution [50°; see step 1 in this section and (a) in the following section] to the microbead layer [see (b) in the following section]. Repeat this process for each sample. 4. When the agarose "glue" has set (1 min), place the comb in the gel mold with the sample facing in the direction the DNA is to be electrophoresed. Pour the agarose gently into the mold and allowed it to thoroughly cool (0.5 hr). To avoid disturbing the microbead layer when the comb is removed, push the comb gently but firmly away from the embedded microbeads (until a small space is visible between the side of each well and tooth) and then pull straight up. 5. Run the gel as usual for PFGE. N o t e s on Pulsed-Field Gel Electrophoresis
a. The 0.5% agarose solution, which will serve as a "glue" to hold the microbeads on the comb, should not be above 55° when it is used or it will melt microbeads made from LMP agarose.
20
ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A
[3]
b. In step 3, the agarose replaces the TE that has been blotted off and the microbead layer will again appear smooth. The agarose should be added in droplets from the top and bottom edges of the microbead mass to avoid displacing the microbeads. Do not allow the microbeads to dry on the comb before the agarose "glue" is added, as this will adversely affect the quality of the pulsed-field gel. c. We have found that loading microbeads on the comb gives the best results for PFGE. As an alternative to this method, the microbeads can be loaded directly into the wells after the gel has set and then sealed in place with agarose. However, we typically have a higher background, more diffuse bands, and more problems with "streaking" when we use this latter approach. Concluding Remarks High-quality, intact genomic DNA can be rapidly prepared and digested in agarose microbeads with the protocols described here. Complete lysis and deproteinization are achieved with a combined incubation time of 2 hr or less. Furthermore, digestion with all restriction enzymes tested has been complete within 1 hr, the time typically allowed for the digestion of DNA in solution. Microbeads not only protect large DNA molecules from shear, but also act as giant DNA-carrying "cells," thus converting DNA into a "solid state" and allowing its easy and rapid transfer to various solutions by sedimenting, washing, and resuspending the microbeads. In addition to E. coli and S. cerevisiae, we have successfully applied these microbead protocols to Pseudomonas aeruginosa, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Plasmodium species, and Magnaporthe grisea and we are confident that they can be adapted to most, if not all, types of cells.
[3] I s o l a t i o n o f L o w M o l e c u l a r W e i g h t D N A f r o m B a c t e r i a a n d A n i m a l Cells
By
SHRIKANT ANANT and
KIRANUR N. SUBRAMANIAN
Introduction Procedures are described for the rapid isolation of low molecular weight DNAs, such as plasmids from bacteria and animal cells. Plasmids METHODS IN ENZYMOLOGY, VOL. 216
Copyright © 1992by AcademicPress, Inc. All rights of reproduction in any form reserved.
PREPARING
AND USING AGAROSE MICROBEADS
13
[2] P r e p a r i n g a n d U s i n g Agarose Microbeads B y MICHAEL KOOB and WACLAW SZYBALSKI
Introduction Protocols for preparing and manipulating large DNA molecules without breakage are required for the routine analysis of genomes by pulsed-field gel electrophoresis (PFGE). To protect genomic DNA from shear, cells are typically embedded in agarose blocks before lysis and deproteinization. 1A less commonly used alternative means of preparing stable genomic DNA was devised by Cook to study the chromatin structure of human chromosomes. 2,3 This process starts with whole cells being embedded in agarose microbeads,4'5 which are essentially minute agarose blocks, and is followed by cell lysis and release of DNA, which remains trapped in the microbeads. Several adaptations of Cook's original microbead procedure to pulsedfield gel applications have been described. 6-8 Because of their small size, agarose microbeads potentially offer several advantages over agarose blocks, both in ease of handling and in the speed with which the DNA can be prepared and enzymatically manipulated. Despite this, however, microbeads have not been widely considered to be satisfactory replacements for agarose plugs. This is due for the most part to difficulties many researchers have reported when working with microbead-embedded DNA. Once washed, agarose microbeads tend to stick to plasticware, pipette tips, and to each other, thus complicating manipulations. When loaded in wells and electrophoresed, they often produce bands that are diffuse or streaked. Finally, when cells are embedded at too high a density, trapping and background smearing tend to occur more frequently as compared with DNA embedded in agarose plugs. In the course of our work to develop new genomic cleavage techniques, we have developed a comprehensive, simple set of procedures for prepar1 D. C. Schwartz and C. R. Cantor, Cell (Cambridge, Mass.) 37, 67 (1984). 2 p. R. Cook, EMBO J. 3, 1837 (1984). 3 D. A. Jackson and P. R. Cook, EMBO J. 4, 913 (1985). 4 K. Nilsson, W. Scheirer, O. W. Merten, L. Ostberg, E. Liehl, H. W. D. Katinger, and K. Mosbach, Nature (London) 302, 629 (1983). 5 K. Nilsson, W. Scheirer, H. W. D. Katinger, and K. Mosbach, this series, Vol. 121, p. 352. 6 G. F. Carle and M. V. Olson, this series, Vol. 155, p. 468. 7 M. McClelland, this series, Vol. 155, p. 22. 8 p. j. Piggot and C. A. M. Curtis, J. Bacteriol. 169, 1260 (1987).
METHODS IN ENZYMOLOGY, VOL. 216
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A
[2]
ing and enzymatically manipulating genomic DNA in agarose microbeads that reproducibly overcome the major problems typically associated with these techniques. 9 Incubation steps for lysis, deproteinization, and enzymatic modification are short, the microbeads are easy to handle, and the quality of the resulting pulsed-field gels is usually superior to those typically obtained with agarose plugs. Although the details in the protocol given are for the preparation of Escherichia coli and Saccharomyces cerevisiae genomic DNA, these procedures can be readily applied to most types of cells. Adaptation of the appropriate steps is discussed. Similarly, the protocol for the enzymatic digestion of the microbead-embedded DNA can be easily adapted to almost any other enzymatic modification (e.g., methylationg).
Preparing and Using Agarose Microbeads Solutions Escherichia coil lysis buffer (ELB): Prepare a buffer containing 1 M
NaCI, 0.1 M ethylenediaminetetraacetic acid (EDTA; pH 8.0/25°), 10 mM Tris-HCl (pH 8.0/25°), and 1% (w/v) sodium N-lauroylsarcosine with sterile stock solutions and store at room temperature. Add 1 mg lysozyme (Sigma, St. Louis, MO) and 2 ~1 RNase (10 mg/ml DNasefree stock, stored at 4 °) per milliliter of lysis buffer just before use Yeast spheroplast buffer (YSB): Prepare a solution of three parts sterile SCE (shown below) and two parts sterile 0.5 M EDTA (pH 8.0/25 °) and store at room temperature. Resuspend microbead-embedded yeast in 4 ml of this buffer and just before incubation at 37° add 1 mg lyticase (about 1000 units; Sigma) and 200 ~1 2-mercaptoethanol (about 14.4 M; Sigma) to this suspension. (The Corex tube should be covered with Parafilm during incubation because of the 2-mercaptoethanol) ES: Add 1% (w/v) sodium N-lauroylsarcosine to sterile 0.5 M EDTA (pH 8/25 °) and store at room temperature ESP: Add 1 mg proteinase K (Sigma) per milliliter ES and incubate at 37 ° for 0.5 hr to eliminate DNases. Prepare this solution just before use SCE: Prepare a buffer consisting of 1.0 M sorbitol, 0.1 M sodium citrate, and 60 mM EDTA, adjust to pH 7.0/25 ° with HC1, sterilize, and store at room temperature TE: I0 mM Tris-HCl, 1 mM EDTA, pH 8.0/25 ° 9 M. Koob and W. Szybalski, Science 250, 271 (1990).
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PREPARING AND USING AGAROSE MICROBEADS
15
STE: Dilute sterile 5 M NaC1 stock to 1 M TE PMSF/TE: Prepare a 100 mM phenylmethylsulfonyl fluoride (PMSF) stock by dissolving 26 mg PMSF in 1.5 ml ethanol and store at - 20°. Dilute to 1 mM with TE just before use. (PMSF is toxic and should be handled with care) TEX, 0.1EX, and 0.5EX: Add 0.01% (v/v) Triton X-100 (Sigma) to sterile TE, 0.1 M EDTA (pH 8.0/25°), and 0.5 M EDTA (pH 8.0/25°), respectively, and store at room temperature
Cell Preparation Cells are grown and prepared for embedding in microbeads in essentially the same manner as for agarose plugs. In every case they are washed once in a buffer in which they are stable and then concentrated to twice the final desired concentration in the same buffer. Escherichia coli. Inoculate 50 ml LB (5 g yeast extract, 10 g tryptone, 10 g NaCI, distilled water to 1 liter) with 1 ml overnight culture and shake at 37° until the OD550 is 0.2-0.3. Add chloramphenicol to a final concentration of 180 ~g/ml to stop chromosomal replication and aid in cell lysis. Again incubate at 37° until growth has stopped (0.5-1 hr). Chill on ice, determine the final OD550, and calculate the number of cells per milliliter by assuming that there are 1 × 108 cells/ml at an OD550of 0.24. Concentrate 1-2 × 10 9 cells by centrifugation, wash once with 5 ml STE, and resuspend in STE to a final volume of 2 ml. (NotI-digested DNA from microbeads made with 1 × 109 cells will give fine, light DNA bands on PFGE, and that from microbeads made with 2 × 10 9 cells will give intense, heavy bands.) Saccharomyces cerevisiae. Grow cells by inoculating 10 ml YPD (10 g yeast extract, 20 g peptone, 20 g glucose, distilled water to 1 liter) with a fresh culture and shake overnight at 30°. Wash the cells once with 5 ml SCE and resuspend in SCE to a final volume of 2 ml.
Embedding Cells in Microbeads 1. Add 5 ml of paraffin oil (or light mineral oil) to a sterile 25-ml Pyrex flask and warm to 42 °. Prepare a 1% (w/v) solution of low-melting-point (LMP) agarose (e.g., InCert agarose; FMC, Rockland, ME) in glass-distilled sterile H20 and cool to 50 ° [see (a) in the following section]. 2. Warm the 2 ml of washed cells to room temperature and add them to 2 ml 50° agarose. Swirl briefly to mix, and pour into the warm paraffin oil. 3. Vortex the oil/agarose vigorously [see (b) in the following section] until a fine, milky emulsion has formed (30-60 sec). Immediately swirl the
16
ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A
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flask in an ice/salt water bath (1 min) to cool the oil quickly and solidify the agarose beads. 4. To remove the oil, add 5 ml 0.1EX to the oil/microbeads emulsion, vigorously swirl the flask, and pour the suspension into a 15-ml Corex centrifuge tube. Pellet the microbeads by centrifugation (5 min at 5000 rpm in a Sorvall (Norwalk, CT) SS-34 rotor, 4°). Carefully pour the oil and buffer off the pellet.
Notes on Embedding Cells a. When enzymatic reactions will be performed that require incubation above 55 °, a highly purified normal-melting-point agarose (e.g., GTG agarose; FMC) may be used in place of LMP agarose in the above procedure. However, when this is done, the temperature of the cells, oil, and agarose should be increased to 37, 50, and 50°, respectively. This keeps the agarose from setting before the microbeads have formed. In addition, deproteinization (below) can be carried out at 65 °. b. Vigorous vortexing and rapid cooling (step 3) are the most critical steps in making uniform preparations of small microbeads. The best results are obtained when the flask is held nearly horizontal with its base pressed firmly to the vibrating nub, causing the liquid to froth violently. To entrap larger cells, such as those from human tissue culture, less vigorous mixing should be used to produce slightly larger microbeads. c. Attempting to embed cells at unusually high concentrations will result in large numbers of cells trapped on the microbead surface and free in solution. This in turn will lead to severe clumping and, following cell lysis, an unusually viscous solution. Discard preparations that show these symptoms and repeat the embedding process at a lower cell concentration.
Cell Lysis and Deproteinization 1. To remove the cell walls from the embedded cells, resuspend the microbead pellet in 4 ml of the appropriate buffer (ELB or Y S B - - n o t e that the lyticase and 2-mercaptoethanol are added only at this point) and incubate at 37° for 0.5 hr (E. coli) or 1 hr (yeast). Pellet the microbeads and pour off the buffer [see (a)-(c) in the following section]. 2. Resuspend the pellet in 4 ml ESP, incubate at 52° for 1 hr, and then remove the ESP [see (c) in the following section]. 3. Wash the deproteinized pellet once with 8 ml TE, twice with 4 ml PMSF/TE, once or twice with 8-10 ml TE, and then once with 2 ml 0.5EX. Take care to remove all of the EX from above the pellet after the last wash. 4. To check the quality of the preparation, draw 200/xl of the micro-
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PREPARING AND USING AGAROSE MICROBEADS
17
beads from the pellet into an uncut Gilson P-200 disposable pipette tip (Rainin, Emeryville, CA) and then expell them back to the pellet. They should pipette smoothly and have a consistency similar to that of a heavy oil. If they do not, they either (1) have clumps of microbeads, or (2) are too dry. To eliminate small clumps of microbeads, repeatedly draw them into and forcefully expell them from the pipette tip until they flow smoothly [see (d) in the following section]. In the case of an overly dry pellet, add enough 0.5EX back to the pellet to allow the microbeads to be pipetted. 5. Cover the Corex tube with Parafilm and store the microbeads at 4° [see (e) in the following section]. Notes on Preparing Genomic DNA a. The enzymes and buffers used in step 1 should be appropriately modified for removing the cell wall from other types of cells. Microbeadembedded cells without cell walls are treated directly with ESP (i.e., a separate lysis step is not necessary). b. For incubation during the lysis and deproteinization steps, the Corex tube containing the resuspended microbeads is placed in the appropriate water bath. Shaking is not necessary. c. To change a buffer, pellet the microbeads by centrifugation (5 min at 5000 rpm in a Sorvall SS-34 rotor, 4°) and pour the buffer carefully off the pellet. Add the new buffer and completely resuspend the pelleted microbeads by vigorous vortexing. Clumps of microbeads that do not separate during vortexing should be broken by pipetting up and down through a disposable transfer pipette. d. In addition to making the microbeads easy to pipette, disrupting microbead clumps allows for more uniform and efficient enzymatic treatment of the DNA in the microbeads and minimizes trapping during pulsedfield gel electrophoresis. The Triton X-100, which does not inhibit most enzymatic reactions at low concentrations, prevents the microbeads from sticking to themselves and to the plastic and glassware with which they came in contact. Incorporation of small amounts of this nonionic detergent in many of the solutions throughout this procedure (as indicated) is critical. e. Although genomic DNA prepared in this way can be stored at 4° and used successfully for over a year, the best results are obtained from microbeads less than a few months old. Enzymatic Digestion of Microbead-Embedded DNA 1. Pipette 20-30/zl of microbeads into a clear or light-colored Eppendoff microfuge tube [see (a) in the following section]. 2. Wash the beads once with 200/zl TEX and then twice with 200/~1
18
ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A
[2]
of the supplier's recommended digestion buffer without bovine serum albumin (BSA) [see (b) in the following section]. 3. Add 0.1 vol BSA (2-3/zl of sterile 1 mg/ml stock solution) and 0.5-1 /xl restriction enzyme to the buffer-equilibrated pellet and mix thoroughly with the end of the pipette tip. Place the microfuge tube in the appropriate water bath and incubate for 1 hr (shaking is not necessary). 4. If the microbeads will be loaded on a gel immediately after the digestion, wash the pellet with 200/zl TEX. For samples that are to be stored for longer than a few hours before loading, add an equal volume of ES to the digest, incubate at 52° for 15 min to inactivate all nucleases completely, and store the sample at 4 °.
Notes on Enzymatic Manipulation of DNA in Microbeads a. Using Gilson P-200 disposable pipette tips, from each of which approximately 5 mm of the end has been removed (use a razor blade), transfer small amounts of microbead suspensions. The larger bore of such pipette tips allows the slow-flowing microbead suspension to be more easily and accurately pipetted. b. To wash the microbead pellet, forcefully pipette TEX or buffer into the tube (the force of the buffer injected into the tube is sufficient to resuspend the microbeads completely), centrifuge for 1 min in a microfuge at maximum speed, and remove the buffer down to the microbead pellet by pipetting with an uncut tip. Although microbeads made from yeast have a slightly milky appearance, those made from E. coli are clear and difficult to see. To distinguish the clear microbead pellet from the buffer, hold the Eppendorf tube up to a light to make the pellet/buffer boundary visible by the difference in diffraction. The buffer remaining in the microbead pellet after the washing process is sufficient for the enzymatic reaction. c. The above protocol can be used for sequential digestions and for enzymatic manipulations other than digesting. In each case, wash the pellet once with TEX (to remove the previous buffer and to coat the microbeads with Triton X-100) and then twice with the appropriate buffer. Add the BSA, enzyme, and other necessary reagents directly to the bufferequilibrated pellet.
Pulsed-Field Gel Electrophoresis (PFGE) 1. Prepare (a) an agarose solution suitable for PFGE in a 250-ml Pyrex Erlenmeyer flask and (b) a small amount of a 0.5% solution of the same agarose and place them in a 50° water bath until needed [see (a) in the following section]. 2. Add 10/xl TE to the TEX-washed microbead pellet and mix with
[2]
19
P R E P A R I N G A N D U S I N G AGAROSE MICROBEADS
COMB
~ 0 ["
~
~
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o ~t~
.o,~ t:~
A~ro~ Immersion ~ Level
. . . . . . .
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,~?~7~7
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the pipette tip. Load the TE/microbead suspension directly on a tooth of the (horizontal) comb using a cut pipette tip [see (a) in the preceding section]. Form a uniform, fiat droplet completely covering the portion of the tooth face that will be immersed in agarose (see Fig. 1). Repeat for each sample. 3. Once all samples have been loaded on the teeth, remove the TE by touching the lower edge of the microbead/TE droplet with the edge of a small piece of absorbent paper towel cut to the width of the tooth. Hold the towel against the edge of the tooth until all of the TE has been absorbed from the sample into the towel (about 15 sec) and the texture of the microbeads is visible. If necessary, stray microbeads may be pushed into place with the paper towel at this time. Add 5-10/~1 of the 0.5% agarose solution [50°; see step 1 in this section and (a) in the following section] to the microbead layer [see (b) in the following section]. Repeat this process for each sample. 4. When the agarose "glue" has set (1 min), place the comb in the gel mold with the sample facing in the direction the DNA is to be electrophoresed. Pour the agarose gently into the mold and allowed it to thoroughly cool (0.5 hr). To avoid disturbing the microbead layer when the comb is removed, push the comb gently but firmly away from the embedded microbeads (until a small space is visible between the side of each well and tooth) and then pull straight up. 5. Run the gel as usual for PFGE. N o t e s on Pulsed-Field Gel Electrophoresis
a. The 0.5% agarose solution, which will serve as a "glue" to hold the microbeads on the comb, should not be above 55° when it is used or it will melt microbeads made from LMP agarose.
20
ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A
[3]
b. In step 3, the agarose replaces the TE that has been blotted off and the microbead layer will again appear smooth. The agarose should be added in droplets from the top and bottom edges of the microbead mass to avoid displacing the microbeads. Do not allow the microbeads to dry on the comb before the agarose "glue" is added, as this will adversely affect the quality of the pulsed-field gel. c. We have found that loading microbeads on the comb gives the best results for PFGE. As an alternative to this method, the microbeads can be loaded directly into the wells after the gel has set and then sealed in place with agarose. However, we typically have a higher background, more diffuse bands, and more problems with "streaking" when we use this latter approach. Concluding Remarks High-quality, intact genomic DNA can be rapidly prepared and digested in agarose microbeads with the protocols described here. Complete lysis and deproteinization are achieved with a combined incubation time of 2 hr or less. Furthermore, digestion with all restriction enzymes tested has been complete within 1 hr, the time typically allowed for the digestion of DNA in solution. Microbeads not only protect large DNA molecules from shear, but also act as giant DNA-carrying "cells," thus converting DNA into a "solid state" and allowing its easy and rapid transfer to various solutions by sedimenting, washing, and resuspending the microbeads. In addition to E. coli and S. cerevisiae, we have successfully applied these microbead protocols to Pseudomonas aeruginosa, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Plasmodium species, and Magnaporthe grisea and we are confident that they can be adapted to most, if not all, types of cells.
[3] I s o l a t i o n o f L o w M o l e c u l a r W e i g h t D N A f r o m B a c t e r i a a n d A n i m a l Cells
By
SHRIKANT ANANT and
KIRANUR N. SUBRAMANIAN
Introduction Procedures are described for the rapid isolation of low molecular weight DNAs, such as plasmids from bacteria and animal cells. Plasmids METHODS IN ENZYMOLOGY, VOL. 216
Copyright © 1992by AcademicPress, Inc. All rights of reproduction in any form reserved.
