Dec. 30, 2024
The primary consideration for plasmid purification is separation of plasmid DNA from the chromosomal DNA and cellular RNA of the host bacteria. A number of methods have been developed to generate a cleared lysate that not only remove protein and lipids, but also efficiently remove contaminating chromosomal DNA while leaving plasmid DNA free in solution. Methods for the preparation of cleared lysates that enrich for plasmid DNA include SDS-alkaline denaturation (22&#;23), salt-SDS precipitation (24) and rapid boiling (25).
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The SDS-alkaline denaturation method, which is used in all Promega plasmid isolation systems, is a popular procedure for purifying plasmid DNA because of its overall versatility and consistency. This technique exploits the difference in denaturation and renaturation characteristics of covalently closed circular plasmid DNA and chromosomal DNA fragments. Under alkaline conditions (at pH 11), both plasmid and chromosomal DNA are efficiently denatured. Rapid neutralization with a high-salt buffer such as potassium acetate in the presence of SDS has two effects that contribute to the overall effectiveness of the method.
First, rapid neutralization causes the chromosomal DNA to base-pair in an intrastrand manner, forming an insoluble aggregate that precipitates out of solution. The covalently closed nature of the circular plasmid DNA promotes interstrand rehybridization, allowing the plasmid to remain in solution. Second, the potassium salt of SDS is insoluble, so the protein and detergent precipitate and aggregate, which assists in the entrapment of the high-molecular-weight chromosomal DNA. Separation of soluble and insoluble material is accomplished by a clearing method (e.g., filtration, magnetic clearing or centrifugation). The soluble plasmid DNA is ready to be further purified. There are several methods available to purify plasmid DNA from cleared lysate. These include:
Bacterial Growth and Culture Conditions
Successful isolation of quality plasmid DNA begins with culture preparation. A number of factors can influence the growth of bacterial cells. Bacterial growth in liquid culture occurs in three phases: 1) a short lag phase in which the bacteria become acclimated to the media and begin to divide; 2) a log phase, characterized by exponential growth in which most strains of E. coli will divide every 20&#;30 minutes; and 3) a stationary phase in which growth slows and eventually stops in response to the lack of nutrients in the medium.
No net increase in biomass will occur in the stationary phase, but plasmid replication will continue for several hours after reaching stationary phase. Most strains of E. coli will reach a concentration of 1.0&#;4.0 × 109 cells/ml of culture at this stage, depending on culture media and aeration conditions. Depending on inoculation size and the size of the culture, stationary phase will be reached in 6&#;8 hours
Aeration and temperature are of critical importance. The culture volume should be less than or equal to 1/4 the volume of the container (e.g., 250ml medium in a 1 liter flask); using 1/10 the container volume (e.g., 100ml medium in a 1,000ml flask) produces optimal results. The culture tube or flask should be placed in an orbital shaker (approximately 250rpm) to ensure adequate aeration (33). Baffled flasks may increase aeration and thus yields of plasmid DNA. Since most strains of E. coli grow best at 37°C, this incubation temperature is recommended unless the strain of interest requires different conditions for optimal growth.Aeration and temperature are of critical importance. The culture volume should be less than or equal to 1/4 the volume of the container (e.g., 250ml medium in a 1 liter flask); using 1/10 the container volume (e.g., 100ml medium in a 1,000ml flask) produces optimal results. The culture tube or flask should be placed in an orbital shaker (approximately 250rpm) to ensure adequate aeration (33). Baffled flasks may increase aeration and thus yields of plasmid DNA. Since most strains of E. coli grow best at 37°C, this incubation temperature is recommended unless the strain of interest requires different conditions for optimal growth.
Different culture media will also have a profound effect on the growth of different bacterial strains. Promega plasmid DNA purification systems are appropriate for bacterial cultures grown in 1X Luria-Bertani (LB) medium. However, use of LB-Miller medium containing more NaCl will produce significantly greater yields and is highly recommended. Richer media such as 2X YT, CIRCLEGROW® or Terrific Broth may be used to increase plasmid yields by increasing the biomass for a given volume of culture.
Keep the biomass in a range acceptable for the plasmid isolation system used, as overloading may result in poor purity and yield of the plasmid DNA (see Biomass Processed for more information). Culture incubation time affects both the yield and quality of plasmid DNA isolated. Bacterial cultures grown to insufficient density will yield relatively low amounts of DNA. Overgrown cultures may result in suboptimal yields and excessive chromosomal DNA contamination due to autolysis of bacterial cells after they have reached stationary phase. We do not recommend the use of cultures grown longer than 18&#;20 hours.
Antibiotic Selection
Most plasmids carry a marker gene for a specific antibiotic resistance. By supplementing the growth medium with the antibiotic of choice, only cells containing the plasmid of interest will propagate. Adding antibiotic to the required concentration will help to maximize plasmid yields. Note that adding too much antibiotic can inhibit growth, and too little may cause a mixed population of bacteria to grow&#;both with and without the plasmid of interest. For more information on optimal antibiotic ranges to use in culture as well as the mechanisms of antibiotic action and resistance, see Table 5 (34).
Table 5. Antibiotic Mode of Action and Mechanism of Resistance.
Antibiotic Mode of Action Mechanism of Resistance Working Conc. Stock Solution Ampicillin (Amp) A derivative of penicillin that kills growing cells by interfering with bacterial cell wall synthesis. The resistance gene (bla) specifies a periplasmic enzyme, β-lactamase, which cleaves the β-lactam ring of the antibiotic. 50&#;125µg/ml 50mg/ml in water Chloramphenicol (Cm) A bacteriostatic agent that interferes with bacterial protein synthesis by binding to the 50S subunit of ribosomes and preventing peptide bond formation. The resistance gene (cat) specifies an acetyltransferase that acetylates, and thereby inactivates, the antibiotic. 20&#;170µg/ml 34mg/ml in ethanol Hygromycin (Hygro) A protein synthesis inhibitor that interferes with 80S ribosome translocation and causes mistranslation. The resistance gene (hph) specifies a phosphotransferase that catalyzes the phosphorylation of the 4-hydroxyl group on the cyclitol ring (hyosamine), thereby producing 7&#;-O-phosphoryl-hygromycin B, which lacks biological activity both in vivo and in vitro. 20&#;200µg/ml 100mg/ml in water Kanamycin (Kan) A bactericidal agent that binds to 70S ribosomes and causes misreading of messenger RNA. The resistance gene (kan) specifies an enzyme (aminoglycoside phosphotransferase) that modifies the antibiotic and prevents its interaction with ribosomes. 30µg/ml 50mg/m in water Neomycin (Neo) A bactericidal agent that blocks protein synthesis by binding to the prokaryotic 70S ribosomal subunit. Expression of the bacterial APH (aminoglycoside phosphotransferase) gene (derived from Tn5). 50µg/ml 25mg/ml in water Tetracycline (Tet) A light-sensitive bacteriostatic agent that prevents bacterial protein synthesis by binding to the 30S subunit of ribosomes. The resistance gene (tet) specifies a protein that modifies the bacterial membrane and prevents transport of the antibiotic into the cell. 10µg/ml in liquid culture; 12.5µg/ml in plates 12.5mg/ml in ethanolRecommended Inoculation Procedures
Pick an isolated colony from a freshly streaked plate (less than 5 days old) and inoculate LB medium containing the required antibiotic(s). Incubation with shaking for 8&#;16 hours at 37°C before harvesting generally results in maximum yields of a high-copy-number plasmid. To achieve a highly reproducible yield, determine the cell density reached in a typical experiment, and grow cultures to this density in each subsequent experiment. Typically, after overnight incubation, the absorbance of a tenfold dilution of the culture at a wavelength of 600nm (A600) with a 1cm path length should range from 0.10&#;0.35.
Using a colony from a freshly streaked plate (less than 5 days old), inoculate 5&#;50ml of LB medium containing the required antibiotic(s). Grow this starter culture from 8 hours to overnight at 37°C. The following day, use this culture to inoculate the larger culture flask containing antibiotic-supplemented medium by diluting the starter culture between 100- to 500-fold (e.g., adding 10ml overnight culture to 1 liter medium). Incubate this secondary culture for 12&#;16 hours before harvesting cells. The A600 of a tenfold dilution of the culture should be 0.10&#;0.35. As with smaller cultures, to achieve a highly reproducible yield, determine the cell density used in a typical experiment and grow cultures to this density in each subsequent experiment.
Harvesting
When harvesting bacteria, follow the conditions outlined in either the Wizard® Plus SV Miniprep DNA Purification System or the PureYield&#; Plasmid Midiprep System protocol. If the recommended centrifugation time or speed is exceeded, the pelleted cells may be more difficult to resuspend. Insufficient centrifugation time or speed may result in incomplete harvesting of cells and loss of starting material. Consult a centrifuge instruction manual for conversion of rpm to g-force. Once the bacteria are pelleted, this is a good stopping point in the purification process. Storing the pellet at &#;20°C results in little loss of plasmid DNA and may enhance lysis.
Factors That Affect Plasmid DNA Quality and Yield
Bacterial Strain Selection
The choice of host bacterial strain can have a significant impact on the quality and yield of DNA using any purification method. We recommend the use of host strains such as DH5α&#;, JM109 (Cat.# L) and XL1-Blue, which contain mutations in the endA gene. E. coli strains that are listed as endA1 contain such mutations.
The endA gene encodes a 12kDa periplasmic protein called endonuclease I. This enzyme is a double-stranded DNase that can copurify with plasmid DNA, thus causing potential degradation. RNA acts as a competitive inhibitor and alters the endonuclease specificity from that of a double-stranded nucleolytic enzyme yielding seven-base oligonucleotides to a nickase that cleaves an average of one time per substrate (35&#;36). The function of endonuclease I is not fully understood, and strains bearing endA1 mutations have no obvious phenotype other than improved stability and yield of plasmid obtained from them.
The expression of endonuclease I has been characterized and was found to be dependent on bacterial growth phase (37). In this study, endonuclease I levels were found to be more than 300 times higher during exponential phase compared to stationary phase. In addition, media compositions that encouraged rapid growth (e.g., high glucose levels and addition of amino acids) resulted in high endonuclease I levels.
Strains that contain the wildtype endonuclease A (endA) gene can yield high-quality, undegraded plasmid DNA if special precautions are used to reduce the probability of nuclease contamination and plasmid degradation (37). Promega has performed a thorough investigation of methods at different points in the purification process to ensure the isolation of high-quality DNA from EndA+ (wildtype) bacterial strains. These include: 1) inclusion of an alkaline protease treatment step that degrades nucleases in the Wizard® Plus SV Minipreps DNA Purification System; 2) optimization of culture conditions to limit in vivo expression during bacterial growth; 3) heat inactivation during and after purification; 4) optimization of protocol conditions to limit binding of the nuclease to the resin and 5) post-purification methods to remove endonuclease. These methods and results are summarized in Schoenfeld et al. (38) and the Wizard® Plus SV Plasmid DNA Purification System Technical Bulletin. Information on genetic markers in bacterial strains can also be found in Ausubel et al. (33) and Sambrook et al. (39).
Plasmid Copy Number
One of the most critical factors affecting the yield of plasmid from a given system is the copy number of the plasmid. Copy number is determined primarily by the region of DNA surrounding and including the origin of replication in the plasmid. This area, known as the replicon, controls replication of plasmid DNA by bacterial enzyme complexes. Plasmids derived from pBR322 (Cat.# D) contain the ColE1 origin of replication from pMB1. This origin of replication is tightly controlled, resulting in approximately 25 copies of the plasmid per bacterial cell (low copy number). Plasmids derived from pUC contain a mutated version of the ColE1 origin of replication, which results in reduced replication control and approximately 200&#;700 plasmid copies per cell (high copy number).
Some plasmids contain the p15A origin of replication, which is considered a low-copy-number origin. The presence of the p15A origin of replication allows for replication of that particular plasmid in conjunction with a plasmid containing the ColE1 origin of replication. A compatibility group is defined as a set of plasmids whose members are unable to coexist in the same bacterial cell. They are incompatible because they cannot be distinguished from one another by the bacterial cell at a stage that is essential for plasmid maintenance. The introduction of a new origin, in the form of a second plasmid of the same compatibility group, mimics the result of replication of the resident plasmid. Thus, any further replication is prevented until after the two plasmids have been segregated to different cells to create the correct prereplication copy number (40). Most plasmids provided by Promega, including the pGEM® Vectors, are considered to be high-copy-number. The only exception is the pALTER®-MAX Vectors.
Some DNA sequences, when inserted into a particular vector, can lower the copy number of the plasmid. Furthermore, large DNA inserts can also reduce plasmid copy number. In many cases, the exact copy number of a particular construct will not be known. However, many of these plasmids are derived from a small number of commonly used parent constructs.
Appropriate Sample Size and Throughput
Depending on the volume of the bacterial culture, there are different isolation systems for your needs. For small-volume bacterial cultures of 0.6&#;3ml, use a system like the PureYield&#; Plasmid Miniprep System (Cat.# A, A), which gives a plasmid DNA yield of 1.5&#;7.5μg with an A260/A280 &#;1.8 from a 0.6ml overnight bacterial culture with a total biomass (O.D.600 of culture × volume of culture in μl) of 1.3&#;8.
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Related links:Figure 16. The Vac-Man® 96 Vacuum Manifold. This 96-well vacuum manifold is used for processing SV 96 plates for plasmid, genomic and PCR product purification.
For larger cultures with volumes ranging from 50&#;100ml, the PureYield&#; Plasmid Midiprep System (Cat.# A, A, A) is a good choice. With this system, a 50ml culture of a high-copy-number plasmid with a total biomass of 100&#;200 O.D.600 units will yield 100&#;200µg of plasmid. The PureYield&#; Plasmid Maxiprep System (Cat.# A, A) can isolate plasmid from 100&#;250ml of culture with yields up to 1mg of plasmid DNA with an A260/A280 >1.7 from 250ml of overnight bacterial culture, transformed with a high-copy-number plasmid.
For high-throughput processing, systems based on a 96-well format can be performed manually with a vacuum manifold (e.g., Vac-Man® 96 Vacuum Manifold; Figure 16) using silica membrane technology such as the Wizard® SV 96 Plasmid DNA Purification System (Cat.# A, A, A). Alternatively, an automated liquid-handling workstation can process multiwell plates with MagneSil® PMPs and a 96-well magnet (e.g., MagnaBot® 96 Magnetic Separation Device; Figure 17) using the Wizard® MagneSil® Plasmid Purification System (Cat.# A, A, A).
Figure 17. The MagnaBot® 96 Magnetic Separation Device. This 96-well magnet is used for capturing MagneSil® PMPs for DNA purification.
Yields for these systems using high-copy-number plasmid range from 3&#;5µg for the Wizard® SV 96 Plasmid DNA Purification System and up to 6µg for the Wizard® MagneSil® Plasmid Purification System. Smaller plasmid amounts are helpful for assessing the success of a cloning experiment by PCR or restriction digestion or for use in a coupled transcription/translation system like the TNT® Quick Coupled Transcription/Translation System (Cat.# L, L).
Optical density (O.D.) is the measure of how much light is blocked by the biomass of the bacterial culture in a path length of 1cm. The density of the culture is measured at a wavelength of 600nm and can have a great effect on plasmid isolation success. For example, the Wizard® SV 96 Plasmid Purification System has a maximum biomass recommendation of 4.0 O.D.600 to avoid clogging of the Wizard® SV 96 Lysate Clearing Plate (Cat.# A, A), so calculating the O.D. of the culture is necessary.
O.D./ml culture = 600nm absorbance reading × dilution factor
For O.D. measurement, a 1:10 dilution is typically used (e.g., 0.1ml culture in 0.9ml culture medium) to keep the reading in the range of 0.1&#;1.0, where the spectrophotometer is most accurate. For the example above, if the 1:10 dilution reading is 0.15, meaning that each milliliter of culture is 1.5 O.D., no more than 2.67ml culture can be processed (4 O.D. divided by 1.5 O.D./ml = 2.67ml). Exceeding the recommendations of the plasmid purification system may cause clogging or contamination of the system.
Many plasmid isolation systems indicate they are transfection-quality (e.g., the PureYield&#; Plasmid Systems or the Wizard MagneSil Tfx&#; System, Cat.# A). This may be important, as some cultured cells are sensitive to the amount of endotoxin and other contaminants present in the plasmid preparation. Endotoxin is a lipopolysaccharide cell wall component of the outer membrane of Gram-negative bacteria (i.e., all E. coli strains) that can copurify with the plasmid DNA regardless of the purification system used. The amount of this molecule varies by bacterial strain, growth conditions and isolation method. In the PureYield&#; Plasmid Systems, there is an Endotoxin Removal Wash solution that reduces the amount of endotoxin, proteins and other contaminants eluted with the plasmid DNA. For many common cell lines, like 293 and HeLa, the amount of endotoxin present for routine transfections has a minimal effect on the efficiency of transfection (41).
Many factors influence transfection efficiency and/or cellular death including the type and amount of transfection reagent, cell confluency, DNA amount and incubation time with the reagent:DNA complex. Each of these factors will need to be optimized for each cell line-plasmid combination transfected in order to minimize cell death and maximize transfection efficiency. In our experience, transfection experiments with HeLa and NIH/3T3 cells demonstrated that there was little DNA preparation difference with four different plasmid isolation systems used (based on silica membrane, anion exchange and silica resin) when comparing efficiencies using the same transfection reagent. However, the transfection reagent used for DNA uptake had a significant effect on transfection efficiency and cell death. For general considerations for optimization, consult our Transfection guide.
Silica Column-Based Systems
Promega products like the Wizard® Plus SV Minipreps DNA Purification System (Cat.# A, A, A) and the PureYield&#; Plasmid Systems combine the benefits of alkaline lysis with the rapid and easy purification by silica. This is done by using a silica-based membrane in a column format to bind the plasmid DNA contained in the cleared alkaline lysates. Purification is based on selective adsorption of DNA to the silica membrane in the presence of high concentrations of chaotropic salts, washes to efficiently remove contaminants, and elution of the DNA with low-salt solutions such as TE buffer or water.
Purified plasmid DNA is used in many applications from preparing vectors for cloning to generating templates for transcription or coupled transcription/translation reactions. The silica-based purification systems from Promega minimize the amount of salts and other impurities carried over during isolation, which can negatively affect downstream applications, lower yield or prevent enzyme systems from synthesizing the product of interest.
PureYield&#; Plasmid Systems
The PureYield&#; Plasmid Systems isolates high-quality plasmid DNA for use in eukaryotic transfection and in vitro expression experiments. The unique reagents, proprietary matrix and silica membrane-based design of the PureYield&#; Systems greatly reduces the amount of time spent on purification compared to silica resin or other membrane-column methods. While the unique Endotoxin Removal Wash removes protein, RNA and endotoxin contaminants from the bound DNA, the Column Wash Solution followed by membrane drying eliminates salts and alcohols from the plasmid prep, allowing the purified plasmid to be used for highly sensitive applications such as transfection, in vitro transcription and coupled in vitro transcription/translation. An additional benefit is that the same degree of purification can be obtained even with low-copy-number plasmids. Although the system works best for plasmids less than 10kb, plasmids as large as 18kb have been purified.The PureYield&#; Plasmid Systems isolates high-quality plasmid DNA for use in eukaryotic transfection and in vitro expression experiments. The unique reagents, proprietary matrix and silica membrane-based design of the PureYield&#; Systems greatly reduces the amount of time spent on purification compared to silica resin or other membrane-column methods. While the unique Endotoxin Removal Wash removes protein, RNA and endotoxin contaminants from the bound DNA, the Column Wash Solution followed by membrane drying eliminates salts and alcohols from the plasmid prep, allowing the purified plasmid to be used for highly sensitive applications such as transfection, in vitro transcription and coupled in vitro transcription/translation. An additional benefit is that the same degree of purification can be obtained even with low-copy-number plasmids. Although the system works best for plasmids less than 10kb, plasmids as large as 18kb have been purified.
The unique combination of reagents in the PureYield&#; Plasmid Miniprep System purifies plasmid either directly from 0.6ml of bacterial culture or cell pellets from up to 3ml of cell culture (Figure 18). A typical overnight culture is grown in LB medium for 16&#;18 hours. If the cell pellet method is chosen, cells are harvested by centrifugation, then resuspended in 600μl of TE buffer or water. Purifying DNA directly from bacterial culture takes less than 10 minutes with elution volumes as low as 30μl, resulting in more concentrated plasmid DNA. The low elution volume is possible because the column design retains virtually no buffer. A transfection comparison of plasmid isolated using the PureYield&#; Plasmid Miniprep System in various cell lines can be found in Figure 19.
Figure 18. The PureYield&#; Plasmid Miniprep System yields transfection-quality DNA in approximately 10 minutes.
To isolate larger quantities of high-quality plasmid DNA, use the PureYield&#; Plasmid Midiprep System. This plasmid midiprep system is designed to purify 100&#;200µg of plasmid DNA with an A260/A280 >1.7 from a 50ml overnight culture of bacteria in as little as 30 minutes, if the culture is grown with a high-copy-number plasmid, reaching a total optical density (O.D.600 of culture × volume of culture) of 100&#;200. Larger volumes up to 250ml can be processed, but require greater volumes of solutions than that supplied with the PureYield&#; Plasmid Midiprep System.
Figure 19. Plasmid DNA prepared using the PureYield&#; Plasmid Miniprep System consistently works well in transfection experiments. The pGL4.13[luc2/SV40] Vector (Cat.# E) was prepared using a competing system or the PureYield&#; Plasmid Miniprep System. Five different commonly used mammalian cell lines were transfected with the plasmid, and transfection efficiency was assessed by measuring the luciferase activity using the ONE-Glo&#; Luciferase Assay System (Cat.# E; n = 6).
The PureYield&#; Plasmid Midiprep System is designed for purification by vacuum using a manifold such as the Vac-Man® Laboratory Vacuum Manifold (Cat.# A), but there are alternative protocols that use all centrifugation or both vacuum and centrifugation. All protocols generate high-quality purified plasmid DNA. A swinging-bucket tabletop centrifuge or the Eluator&#; Vacuum Elution Device (Cat.# A) is required for the final elution step regardless of the protocol chosen.
For a larger plasmid isolation capacity, the PureYield&#; Plasmid Maxiprep System is able to purify up to 1mg of plasmid DNA with an A260/A280 >1.7 from 250ml of overnight bacterial culture, transformed with a high-copy-number plasmid in approximately 60 minutes. As with the midiprep system, the protocol requires a vacuum pump and manifold (e.g., the Vac-Man® Laboratory Vacuum Manifold, 20-sample), a centrifuge with a fixed-angle rotor for lysate clearing and either a tabletop centrifuge with a swinging bucket rotor or the Eluator&#; Vacuum Elution Device for the final elution step.
High-quality, purified plasmids are used for automated fluorescent DNA sequencing as well as for other standard molecular biology techniques including restriction enzyme digestion and PCR. Whether you are isolating a few samples or a 96-well plate, there is a silica membrane-based system available.
For manual purification, the Wizard® Plus SV Minipreps DNA Purification System provides a simple and reliable method for rapid isolation of plasmid DNA using a column-based silica membrane (see Figure 20 for overview of method). The entire miniprep procedure can be completed in 30 minutes or less, depending on the number of samples processed. The plasmid DNA from 1&#;10ml of overnight E. coli culture can be purified by using either a vacuum manifold like the Vac-Man® Laboratory Vacuum Manifold (process up to 20 samples) or a microcentrifuge (number of samples processed depends on rotor size).
This system can be used to isolate any plasmid hosted in E. coli but works most efficiently when the plasmid is less than 20,000bp in size. The yield of plasmid will vary depending on a number of factors, including the volume of bacterial culture, plasmid copy number, type of culture medium and the bacterial strain used as discussed in Factors that Affect Plasmid DNA Quality and Yield. The DNA binding capacity of the SV membrane is up to 20µg of high-quality plasmid DNA. An alkaline protease treatment step in the isolation procedure improves plasmid quality by digesting proteins like endonuclease I.
Figure 20. Overview of the Wizard® Plus SV Minipreps DNA Purification System centrifugation protocol.
Paramagnetic beads are added to the sample, and their surfaces attract nucleic acids to bind to the beads. Using a strong magnet, the beads are held in place while removing unwanted material. After washing, the genetic material is eluted from the beads in water or a low-salt buffer. Using Dynabeads magnetic beads, many separation applications can be achieved.
Magnetic bead isolation is now one of the most popular nucleic acid extraction methods due to its scalability and automation compatibility. MagMAX bead kits and KingFisher Sample Purification Systems are designed to work together to efficiently purify a variety of nucleic acids.
If you are looking for more details, kindly visit Nucleic Acid Purification Kit.
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