what does silica resin do in dna extraction

0000067201 00000 n We investigate the DNA-silica binding mechanism using molecular dynamics simulations. Implementing automated nucleic acid purification technologies onto your high-throughput workflow can be challenging and time-consuming. This automated protocol also can be adapted to other robotic workstations. This method relies on the fact that nucleic acid will bind to the solid phase of silica under certain conditions. Optimization of extraction methodologies is key for success with challenging sample types and demanding downstream applications. 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. Related content In From the smallest bones come the biggest secrets read about the work of former University of Otago Masters student Lachie Scarsbrook. Under alkaline conditions (at pH 11), both plasmid and chromosomal DNA are efficiently denatured. With the target material bound, the flow-through can be removed. In many protocols, a combination of chemical disruption and another is often used since chemical disruption of cells rapidly inactivates proteins, including nucleases. The Plate Clamp 96 (Cat.# V8251) is recommended for automated protocols and is designed to ensure PCR plates are uniformly flat for liquid transfer on a robotic platform. Toxic and mutagenic substances such as phenol, chloroform, and ethidium bromide are also not required. A vacuum manifold or a microcentrifuge is used for sample processing. Nucleic acid binds to cellulose in the presence of high salt and alcohols. This chemistry can be adapted to either paramagnetic particles (PMPs), like Promega silica-coated MagneSil PMPs, or silica membrane column-based formats. Jiang X, Liu X, Yu Q, Shen W, Mei X, Tian H, Wu C. Mater Today Bio. Davies, J. and Smith, D.I. We have developed procedures for use on several robotic workstations with standard 96- and 384-well amplification plates. Rapid and simple method for purification of nucleic acids. PubMedGoogle Scholar. The salt concentration and pH conditions of the buffers used determine whether DNA is bound or eluted from the column. For small-volume bacterial cultures of 0.63ml, use a system like the PureYieldPlasmid Miniprep System (Cat.# A1223, A1222), which gives a plasmid DNA yield of 1.57.5g 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 l) of 1.38. The resulting purified DNA is ready to use in downstream applications, including amplification assays. 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. 1990 Mar;28(3):495-503. 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). Figure 7. These include both membrane-based systems (e.g., the single-column Wizard SV Genomic DNA Purification System (Cat.# A2360, A2361) or the high-throughput, 96-well Wizard SV 96 Genomic DNA Purification System (Cat.# A2370, A2371) and easily automated paramagnetic silica systems. However, DNA is not the only molecule that can absorb UV light at 260nm. As a guideline, the A260/A230 is best if greater than 1.5. The MagneSil PMPs are considered a mobile solid phase with binding of nucleic acids occurring in solution. The FFPE Plus chemistry is designed to provide high yield of DNA from FFPE when measured by spectroscopy that is suitable for amplification applications including qPCR, multiplex PCR and NGS. The Wizard MagneSil Tfx System provides a simple and reliable method for the rapid isolation of transfection-quality plasmid DNA in a multi-well format. QIAGEN offers a wide range of specialized nucleic acid purification products based on four highly developed purification technologies: solid-phase, anion-exchange technology; QIAGEN Plasmid Plus technology; silica gel membrane technology; and magnetic particle technology. Chen, C.W. Heating to 65C with the GuHCl lysis solution helps to break down the cell and nuclear membranes, and also denatures enzymes that can degrade the purified DNA. The final step in the DNA extraction protocol is the release of pure DNA or RNA from the silica. Spectrophotometry is a common way to evaluate the quality of extracted DNA and RNA. Storing the pellet at It is based on the principle of binding nucleic acid to immobilized solid-phased spin columns of different materials under specific circumstances. Due to the proprietary binding chemistry, up to 50 g of transfection-grade plasmid DNA per well can be obtained from up to 5 ml of an E. coli culture. For small PCR fragments (<500bp), optimal recovery requires a 95% ethanol wash. For larger fragments (>500bp), optimal results are achieved using an 80% ethanol wash. 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. Designed for BigDye Sanger sequencing reaction cleanup, the Wizard MagneSil Sequencing Reaction Clean-Up System (Cat.# A1831, A1832, A1835) can be placed on a robotic platform and purified using the MagneSil PMPs to clean up sequencing reaction products prior to analysis. 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. To isolate larger quantities of high-quality plasmid DNA, use the PureYield Plasmid Midiprep System. Figure 15. Comparison of standard anion-exchange and QIAGEN anion-exchange resin selectivity:A: Plasmid DNA and RNA co-elute using conventional anion-exchange resin, whileB: QIAGEN anion-exchange resin allows the elution of plasmid DNA and RNA at distinct salt concentrations. This membrane-based system can bind up to 60g of DNA and concentrate as much as 300l of dilute DNA, recovering isolated DNA fragments or PCR products in as little as 10 minutes, depending on the number of samples processed and the protocol used. Once extracted, DNA can be used for molecular analyses including PCR, electrophoresis, sequencing, fingerprinting and cloning. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Spin column-based nucleic acid purification is a solid phase extraction method to quickly purify nucleic acids. Like the other cookies we use, strictly necessary cookies may be either first-party cookies or third - party cookies. The salt concentration and pH conditions of the buffers used determine whether DNA is bound or eluted from the column. It essentially combines the classic Buffer 3 of a plasmid prep, which contains acetic acid to neutralize Buffer 2, as well as guanidinium to get that plasmid DNA to bind to the silica. Huh-7 cells were transfected using 200 ng plasmid DNA and 0.5 l Attractene Transfection Reagent or 300 ng plasmid DNA and 0.75 l Attractene Transfection Reagent. Automation eliminates the hands-on time and labor of manual purification, giving you more time and energy to focus on your research. These latter techniques use nanogram amounts of DNA per reaction. Two hundred microliters of blood was used for genomic DNA purification (n = 3 or 4), and the amount of isolated gDNA was quantitated by absorbance spectroscopy. Uusitalo JJ, Inglfsson HI, Akhshi P, Tieleman DP, Marrink SJ. This system is of technological importance, and also of interest to explore how negatively charged DNA can bind to a silica surface, which is also negatively charged at pH values above its isoelectric point near pH 3. Fast, inexpensive The cells or tissues are digested with Proteinase K in the presence of EDTA to inhibit DNases. Eluting and storing the DNA in TE buffer, for example, is helpful as long as the EDTA does not impact your chosen downstream applications. Avoid the tedious and time-consuming hassle of preprocessing samples, simply add 50250l of your sample directly into well #1 of the cartridges. Buffers, such as MOPS, sodium phosphate, TrisCl and sodium acetate can be used at the indicated pH. 0000006972 00000 n There are five basic steps of DNA extraction that are consistent across all the possible DNA purification chemistries: 1) disruption of the cellular structure to create a lysate, 2) separation of the soluble DNA from cell debris and other insoluble material, 3) binding the DNA of interest to a purification matrix, 4) washing proteins and other contaminants away from the matrix and 5) elution of the DNA. 0000018807 00000 n 0000003215 00000 n Results will vary depending on the degree of cross-linking due to formalin fixation. This guide provides a comprehensive introduction to DNA and RNA purification methods, including the basics of DNA isolation, plasmid growth and nucleic acid quantification. Figure 18. Finally, most qPCR QC assays, such as the ProNex DNA QC Assay (Cat.# NG1004, NG1005) provide internal controls which are used to detect the presence of inhibitors in the sample prior to attempting a more expensive assay. The DNA purified from many of these samples can be used in PCR-based testing for Genetically Modified Organism (GMO) DNA sequences, such as by quantitative analysis using TaqMan assays. A further explanation of how DNA binds to silica is based on the action of guanidinium chloride (GuHCl), which acts as a chaotrope. The average A260/A280 ratios are: SV 96, 1.7 0.08; SV vacuum method, 1.7 0.14; SV spin method, 1.7 0.14. Figure 16. Traditionally, automation refers to the use of large, specialized and costly equipment that requires extensive training to operate and maintain. Most strains of E. coli will reach a concentration of 1.04.0 109 cells/ml of culture at this stage, depending on culture media and aeration conditions. Hoechst bisbenzimidazole dyes or PicoGreen selectively bind double-stranded DNA (dsDNA). Information on genetic markers in bacterial strains can also be found in Ausubel et al. Because ethidium bromide is a known mutagen, precautions need to be taken for its proper use and disposal (43). As a result of the combination of binding capacity and relatively small elution volume, we can get high concentration eluates for nucleic acids. 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). This problem has been solved! It is a colorless (white as in powder form), water-soluble and organic molecule. Elution points of different nucleic acids from QIAGEN Resin as a function of pH and NaCl concentration. This in-turn provides you with high-quality material for different experiments like cloning and long-range sequencing. No net increase in biomass will occur in the stationary phase, but plasmid replication will continue for several hours after reaching stationary phase. QIAGEN resin has different binding capacities for different classes of nucleic acids. (1973) The use of sodium perchlorate in deproteinization during the preparation of nucleic acids. The yield of DNA from this system will vary depending on source type and extent of food processing. Depending on the starting material, typical enzymatic treatments can include: lysozyme, zymolase and liticase, proteinase K, collagenase and lipase, among others. While there are general trends, the DV200 score does not necessarily correlate with success in downstream assays such as qPCR. Copy number is determined primarily by the region of DNA surrounding and including the origin of replication in the plasmid. The DNA is eluted under high salt conditions, and then recovered by ethanol precipitation. Bead-based clearing, like the method used with Promega particle-based plasmid prep kits, can be used in automated protocols, but can be overwhelmed with biomass. The procedures are simple, rapid, involve no organic solvents and do not require multiple tube transfers for most types of samples. Most of these involve purifying DNA by passing it through a column containing a resin that binds DNA but not other cell components. The Maxwell RSC (left) and Maxwell RSC 48 (right). Please try again or contact Customer Service. Chaotropic salts present in high quantities are able to disrupt cells, deactivate nucleases and allow nucleic acid to bind to silica. Separation of nucleic acids at neutral pH on anion-exchange resins. K. A. Melzak, C. S. Sherwood, R. F. B. Turner, C. A. Haynes. Purification on QIAGEN resin is based on the interaction between negatively charged phosphates of the nucleic acid backbone and positively charged DEAE groups on the surface of the resin (see figure Binding principle of QIAGEN resin). Tan, S. Y. This DNA purification guide addresses general information on the basics of DNA extraction, plasmid preparation and DNA quantitation, as well as how optimized purification techniques can help increase your productivity, so you spend less time purifying DNA and more time developing experiments and analyzing data. BioTechniques, 54(3). 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. The silica method in particular has been shown to be robust when extracting DNA from forensic samples [1]; it is also amenable to automation [2, 3]. After that, you will need to contact Customer Service to unlock your account. Remove any extra proteins and other contaminants from the mixture by centrifugation. Efficiency of DNA isolation methods based on silica columns and magnetic separation tested for the detection of Mycobacterium avium Subsp. A verified email address is required to access the full functionality of your Promega.com account. Each point is the mean of n=4 values with error bars of 1 standard deviation. The innovative binding buffer included in kits ensures very specific binding conditions, providing DNA quality that is comparable to anion-exchange preps. The soluble plasmid DNA is ready to be further purified. The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Table 8. Start lysis right away and let the samples thaw upon lysis incubation. Utilizing spin, vacuum or magnetic-based methods, our manual single-prep solutions are best for processing less than 24 samples at a time. Looking for extraction options by sample scale or type? 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. After sample addition, the Maxwell RSC moves the paramagnetic particles and associated nucleic acids through multiple steps ultimately yielding highly pure RNA or DNA in 30100l. The basic principle of DNA/RNA extraction. Small-to large-scale plasmid purification, High-molecular-weight genomic DNA isolation, QIAGEN Plasmid Plus 96 Miniprep and BioRobot Kits, DNA isolation from animal tissues and cells, DNA and RNA isolation from various samples, Transfection into most cell lines (including sensitive cell lines such as Huh-7), Preparation of short hairpin vectors (sh-vectors). When sufficient growth has been achieved, the cells are pelleted by centrifugation to remove them from the growth medium. Specialized, sample-type specific purification kits may be needed for more complex and challenging samples that contain degraded DNA or a have low concentrations of DNA. The Maxwell RSC FFPE Plus DNA method has been observed to produce more yield by absorbance and fluorescence, while the Maxwell RSC DNA FFPE method produces more yield by PCR. 0000046283 00000 n One microliter of purified genomic DNA was amplified using PCR Master Mix (Cat.# M7502) and mouse-specific IL-1 primers (1.2kb product). These methods and results are summarized in Schoenfeld et al. You can set your browser to block or alert you about these cookies, but some parts of our services will not work without them. The Maxwell RSC Buffy Coat DNA Kit (Cat.# AS1540) provides a simple, automated method of genomic DNA extraction using the convenient, prefilled cartridge format of the Maxwell RSC Instrument. Spin columns contain a silica resin that selectively binds DNA, depending on the salt conditions and other factors influenced by the extraction method. This membrane-based system, which can bind up to 40g DNA, allows recovery of isolated DNA fragments or PCR products in as little as 20 minutes, depending on the number of samples processed and the protocol used. The ReliaPrep Blood gDNA Miniprep System processes 200l of blood or body fluid, either fresh or frozen, in less than 40 minutes. DNA is more stable at a slightly basic pH and will dissolve faster in a buffer. The DNA IQ System uses a silica-based paramagnetic resin to isolate DNA from liquid samples and samples on solid supports. The preprogrammed methods control the heating, shaking, magnetization and timing of the steps required for the semi-automated purification. In addition, media compositions that encouraged rapid growth (e.g., high glucose levels and addition of amino acids) resulted in high endonuclease I levels. Alternatively, you can use TE-4 buffer, which is 10mm Tris-HCl, 0.1mm EDTA (pH 8.0). Application and sample type-focused kits make the Maxwell Instruments a versatile extraction instrument for laboratories that may work with one or all of these different applications. Overgrown cultures may result in suboptimal yields and excessive chromosomal DNA contamination due to autolysis of bacterial cells after they have reached stationary phase. Forensic Science International: Genetics, 44, 102191. Usually clearing is accomplished by centrifugation, filtration or bead-based methods. We use these cookies to ensure our site functions securely and properly; they are necessary for our services to function and cannot be switched off in our systems. However, the automated operation of spin columns is required to connect with the Internet of things to develop a next-generation advanced intelligent separation system. The covalently closed nature of the circular plasmid DNA promotes interstrand rehybridization, allowing the plasmid to remain in solution. The most common purity calculation is determining the ratio of the absorbance at 260nm divided by the reading at 280nm. https://doi.org/10.1016/b978-0-12-802971-8.00021-3. Plasmid DNA prepared with QIAGEN Plasmid Plus 96 Miniprep and BioRobot Kits resulted in highly efficient transfection into sensitive cell lines. A bactericidal agent that binds to 70S ribosomes and causes misreading of messenger RNA. For example, when the same samples were quantitated by qPCR assays of various targets and fragment sizes, the yield by qPCR does not correlate well with the DV200 scores. Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J. This means that if the A260 number is used for calculation of yield, the DNA quantity may be overestimated (43). That first extraction led to the simple discovery that a material exists within cells that precipitates out of acidic solution and dissolves into alkaline solution. Journal of Membrane Science, 311(12), 336348. HiSpeed Midi Tips, provided in the HiSpeed Plasmid Midi Kit, contain a newly developed anion-exchange resin. DNA and RNA Isolation Techniques for Non-Experts, https://doi.org/10.1007/978-3-030-94230-4_5, Techniques in Life Science and Biomedicine for the Non-Expert, Tax calculation will be finalised during checkout. Whole blood was obtained from several individuals, and white cell counts were determined using a hemocytometer. Clipboard, Search History, and several other advanced features are temporarily unavailable. (1962) The effect of electrolytes on the stability of the deoxyribonucleate helix. The high concentration of salt causes the proteins to fall out of solution, and then centrifugation separates the soluble nucleic acid from the cell debris and precipitated protein (1). and Quigley, M. (1981) A rapid boiling method for the preparation of bacterial plasmids. Fast, inexpensive The Maxwell Systems purify samples using paramagnetic particles (PMPs), which provide a mobile solid phase that optimizes sample capture, washing and elution of the nucleic acid. In this study, endonuclease I levels were found to be more than 300 times higher during exponential phase compared to stationary phase. https://doi.org/10.1186/s12575-018-0077-6, Walsh, P. S., Metzger, D. A., & Higuchi, R. (2013). The PureLink Genomic DNA Purification Kit is based on the selective binding of DNA to silica-based membrane in the presence of chaotropic salts. In terms of sensitivity in nucleic acid detection, it is surpassed only by ddPCR. 0000003710 00000 n This convenient protocol is designed for the manual purification of DNA from a variety of food samples including corn seeds, cornmeal, soybeans, soy flour and soy milk, generating results in one-third of the time of traditional methods. Procedure [ edit] 0000006704 00000 n 60ada`f6 FfLgR`K_@ 6p. Privacy Policy The chemical formula of EDTA is C 10 H 16 N 2 O 8. 0000003261 00000 n from the cells. (1991) Precipitation of DNA by polyethylene glycol and ethanol. Conditions can be adjusted to preferentially bind different species and sizes of nucleic acid. 0000010296 00000 n Silica based salting out is faster and more efficient than traditional salting out methods. The cell consists of a cell wall/cell membrane and cytoplasm, where . Compare plasmid DNA prep kits to find the purification solution that is right for you. The Maxwell HT DNA FFPE Isolation System purifies nucleic acid using paramagnetic particles, which provide a mobile solid phase to optimize binding, washing and purification of gDNA. The yield of genomic DNA from the ReliaPrep Blood gDNA Miniprep System varies with white blood cell count. Since plant materials can be particularly challenging to lyse, especially when working with tough or woody tissues, additional required equipment includes not only a magnet (MagnaBot FLEX 96 Magnetic Separation Device, Cat.# VA1920) but also a device capable of breaking up seed or leaf material (e.g., Geno/Grinder 2000 from SPEX CertiPrep, Inc.). 0000002929 00000 n When selecting your elution buffer, it is important to consider the requirements of your desired downstream processes. The data were processed . This step may be improved with salt, pH, time, or heat. Google Scholar, McKiernan, H., & Danielson, P. (2017). Please contact Customer Service to unlock your account. Silica resins have been used for DNA and RNA preparation for decades 14,15,16, . No silica-slurry More recently, Promega has commercialized DNA isolation methods that use a cellulose-based matrix. Unable to load your collection due to an error, Unable to load your delegates due to an error. While the latter make use of DNA-adsorbing materials (e.g. 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.# E6110; n = 6). There was an error processing your request. The use of paramagnetic particles for DNA isolation eliminates the need for centrifugation or vacuum manifolds, making the system suitable for full automation. Google Scholar. 0000018594 00000 n DNA/RNA extraction can be divided into two steps: cell lysis and purification. Cooperative effects at water-crystalline silica interfaces strengthen surface silanol hydrogen bonding. The remaining tissue is discarded. Automating reagents onto instrumentation requires a carefully planned and executed approach. government site. The insoluble DNA is then pelleted and separated from salt, isopropanol and RNA fragments via centrifugation. You have not verified your email address. HHS Vulnerability Disclosure, Help Different culture media will also have a profound effect on the growth of different bacterial strains. Spin Column-Based Isolation of Nucleic Acid. Please enable it to take advantage of the complete set of features! 2011 Oct;11(10):8457-68. doi: 10.1166/jnn.2011.4994. However, use of LB-Miller medium containing more NaCl will produce significantly greater yields and is highly recommended. In: DNA and RNA Isolation Techniques for Non-Experts. For larger cultures with volumes ranging from 50100ml, the PureYield Plasmid Midiprep System (Cat.# A2492, A2495, A2496) is a good choice. The most common technique to determine DNA yield and purity is also the easiest methodabsorbance. Other methods of DNA purification involve columns of various sorts, which are packed with ion exchange, or silica based resins or matrices. The total DNA concentration was assessed using the QuantiFluor ONE dsDNA System. The addition of a chaotropic salt, for example 6-m guanidine thiocyanate [9] or 6-m sodium chloride, during or after cell lysis, disrupts the protein structure by interfering with hydrogen bonding, Van der Waals interactions, and the hydrophobic interactions. While both methods generally represent a good balance of yield and purity, the silica membrane column format is more convenient. Current nucleic acid extraction methods and their implications to point-of-care diagnostics. To increase the yield from the Wizard Magnetic 96 DNA Plant System, a scale up in volume with up to 5 leaf punches can be used [as demonstrated in Promega Notes 79]. While the dyes bind preferentially to dsDNA, RNA and nucleotides may contribute to the signal. 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. The sample in binding solution is then transferred to a spin column, and the column is put either in a centrifuge or attached to a vacuum. Promega Corporation (2002) Genomic DNA purification from blood. Learn about the advantages and disadvantages of current DNA/RNA quantitation methods, including absorbance, fluorescent nucleic acid-binding dyes and qPCR. Your purified DNA is ready for analysis in about 50 minutes, and can be used directly in various downstream applications, such as agarose gel electrophoresis. They include a silica resin that selectively binds DNA or RNA relying on the factors involved in the extraction method. Depending on the target material, this can include the use of detergent or other buffers, proteinases or other enzymes, heating to various times/temperatures, or mechanical disruption such as cutting with a knife or homogenizer, using a mortar and pestle, or bead-beating with a bead mill. The DNA can then be washed with high salt and ethanol, and ultimately eluted with low salt. (Vac-Man 96 Vacuum Manifold, Cat.# A2291) and a vacuum pump capable of generating 1520 inches of mercury or the equivalent. For others, we wont set them unless you accept them. Regardless of the system chosen, Promega genomic DNA purification kits provide the required yields of high-quality DNA with minimal contaminants. The Kit is used with the Maxwell RSC and RSC 48 Instruments and can purify DNA from raw and processed food samples, including corn, soybeans, canola, ground beef and ground pork. We can build design features into these chemistries by manipulating the binding conditions to enrich for different categories of nucleic acid (e.g., chemistries that selectively bind RNA versus DNA or large versus small fragments). A single reagent stream is used for all three procedures, making the system both fast and easy. Dash, H. S. (2020). For example, if a 2l sample of undiluted DNA loaded on the gel has the same approximate intensity as the 100ng standard, then the solution concentration is 50ng/l (100ng divided by 2l). When considering FFPE samples, it is important to note that dye-based quantitation does not estimate the integrity of the DNA/RNA or the extent of cross-linking in the sample, which could affect success in downstream assays. 0000005059 00000 n The mechanism behind DNA adsorption onto silica is not fully understood; one possible explanation involves reduction of the silica surface's negative charge due to the high ionic strength of the buffer. A common method of physical disruption is freezing and grinding samples with a mortar and pestle under liquid nitrogen to provide a powdered material that is then exposed to chemical or enzymatic lysis conditions. Affinity Chromatography: This uses silica resins. BioMed Research International, 2009, 574398. Deviations from the appropriate pH values of the buffers at a given salt concentration may result in losses of the desired nucleic acid. 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).

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