How can dna be isolated

Explore our collection of protocols for manual and automated DNA or RNA extraction from a variety of food and plant samples. 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. 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. The soluble plasmid DNA is ready to be further purified.

There are several methods available to purify plasmid DNA from cleared lysate. These include:. 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.

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. Depending on inoculation size and the size of the culture, stationary phase will be reached in 6—8 hours.

Different culture media will also have a profound effect on the growth of different bacterial strains. However, use of LB-Miller medium containing more NaCl will produce significantly greater yields and is highly recommended. 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.

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 Pick an isolated colony from a freshly streaked plate less than 5 days old and inoculate LB medium containing the required antibiotic s.

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 nm A with a 1cm path length should range from 0.

Using a colony from a freshly streaked plate less than 5 days old , inoculate 5—50ml of LB medium containing the required antibiotic s. The following day, use this culture to inoculate the larger culture flask containing antibiotic-supplemented medium by diluting the starter culture between to fold e.

Incubate this secondary culture for 12—16 hours before harvesting cells. The A of a tenfold dilution of the culture should be 0. 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.

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.

The choice of host bacterial strain can have a significant impact on the quality and yield of DNA using any purification method. L and XL1-Blue, which contain mutations in the endA gene. 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— The function of endonuclease I is not fully understood, and strains bearing end A1 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 In this study, endonuclease I levels were found to be more than times higher during exponential phase compared to stationary phase. In addition, media compositions that encouraged rapid growth e. 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 These methods and results are summarized in Schoenfeld et al.

Information on genetic markers in bacterial strains can also be found in Ausubel et al. 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 pBR 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 — 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 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.

Depending on the volume of the bacterial culture, there are different isolation systems for your needs. For small-volume bacterial cultures of 0. A , A , which gives a plasmid DNA yield of 1. This well vacuum manifold is used for processing SV 96 plates for plasmid, genomic and PCR product purification. A , A , A is a good choice. With this system, a 50ml culture of a high-copy-number plasmid with a total biomass of — O. For high-throughput processing, systems based on a well format can be performed manually with a vacuum manifold e.

A , A , A L , L Optical density O. The density of the culture is measured at a wavelength of nm and can have a great effect on plasmid isolation success. A , A , so calculating the O. For O. For the example above, if the dilution reading is 0. 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.

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. The amount of this molecule varies by bacterial strain, growth conditions and isolation method.

For many common cell lines, like and HeLa, the amount of endotoxin present for routine transfections has a minimal effect on the efficiency of transfection 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.

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.

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.

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. A typical overnight culture is grown in LB medium for 16—18 hours. The low elution volume is possible because the column design retains virtually no buffer. The pGL4. A , but there are alternative protocols that use all centrifugation or both vacuum and centrifugation.

All protocols generate high-quality purified plasmid DNA. A is required for the final elution step regardless of the protocol chosen. As with the midiprep system, the protocol requires a vacuum pump and manifold e. 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 well plate, there is a silica membrane-based system available. 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. This system can be used to isolate any plasmid hosted in E. 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.

An alkaline protease treatment step in the isolation procedure improves plasmid quality by digesting proteins like endonuclease I.

These high-throughput systems provide a simple and reliable method for the rapid isolation of plasmid DNA using a silica-membrane well plate. A single plate can be processed in 60 minutes or less. The particles are also completely resuspended during the wash steps of a purification protocol, enhancing the removal of impurities from the DNA.

The protocol also requires a multiwell plate shaker. DNA purified with using this system is greatly reduced in chemical contaminants as well as RNA, protein, and endotoxin, providing high-quality plasmid DNA suitable for transfection, as well as for other standard molecular biology techniques. In addition, a proprietary paramagnetic endotoxin removal resin reduces the level of endotoxin present in the purified plasmid DNA.

The procedure requires no manual intervention and takes approximately 45 minutes to process a single well plate. This automated protocol also can be adapted to other robotic workstations. After a PCR amplification or restriction enzyme digestion, the reaction components include protein and salts that may inhibit subsequent applications and will need to be removed from the DNA fragments.

An agarose gel may be run to isolate a fragment of the correct size if there is more than one product present. Fragment DNA purification can improve efficiency in subsequent reactions. However, nonspecific amplification products and primer dimers can compete for ligation with the desired PCR product, resulting in a low frequency of positive clones.

Additionally, removing the reaction components prior to sequencing will ensure the right primers are used for sequencing reactions and that the fluorescently labeled nucleotides are not competing with the unlabeled dNTPs remaining from the PCR amplification. Applications such as cloning, labeling and sequencing DNA frequently require the purification of DNA fragments from agarose gels or amplification reactions. A , A , A is designed to quickly concentrate and purify dilute DNA solutions, extract and purify DNA fragments of bp—10kb from standard or low-melt agarose gels or to purify products directly from a PCR amplification.

A single reagent stream is used for all three procedures, making the system both fast and easy. Table 6. The system is designed to extract and purify DNA fragments of bp to 10kb from standard or low-melting point agarose or to purify PCR products directly from an amplification reaction, using the SV silica membrane column. This purification kit is a single column system that can be used with a vacuum manifold e. Table 7. Table 8. For direct purification from a reaction, note that any nucleic acid present in solution will be isolated.

Therefore, if an amplification reaction has more than one product, all fragments will be present in the eluted DNA. If you are interested in isolating a single amplicon, separate the reaction products on an agarose gel and cut out the band desired prior to purification.

When purifying DNA from an agarose slice, the primary consideration is to melt the agarose so the DNA is available for binding to the silica membrane. The purified DNA can then be used for cloning or sequencing. The technology is the same as the single-column system, utilizing the SV silica membrane and chaotropic salts to purify the nucleotides and primers from the PCR product s.

This system allows recovery of 96 PCR fragments in as little as 20 minutes in multiwell plate format. Agarose gel analysis. Percent recovery of purified PCR products. Results show the mean and standard deviation for 6 purified fragments of each size. The novel reagent formulation provides significantly improved selectivity, reproducibility and yield relative to traditional dsDNA purification methods.

We have developed procedures for use on several robotic workstations with standard and well amplification plates.

The Plate Clamp 96 Cat. V is recommended for automated protocols and is designed to ensure PCR plates are uniformly flat for liquid transfer on a robotic platform. No user intervention is required from the time the multiwell plates are placed on the robot deck until the samples are loaded onto the DNA sequencer.

DNA yield can be assessed using three different physical methods: absorbance optical density , agarose gel electrophoresis and fluorescent DNA-binding dyes.

Each technique is described below and includes information on necessary accessories e. While all methods are useful, each has caveats to consider when choosing a quantitation approach.

The most common technique to determine DNA yield and purity is also the easiest method—absorbance. All that is needed for measurement is a spectrophotometer equipped with a UV lamp, UV-transparent cuvettes depending on the instrument and a solution of purified DNA. Absorbance readings are performed at nm A where DNA absorbs light most strongly, and the number generated allows one to estimate the concentration of the solution.

To ensure the numbers are useful, the A reading should be between 0. Since RNA also has a great absorbance at nm, and the aromatic amino acids present in protein absorb at nm, both contaminants, if present in the DNA solution, will contribute to the total measurement at nm. Additionally, the presence of guanidine will lead to higher nm absorbance. This means that if the A number is used for calculation of yield, the DNA quantity may be overestimated To evaluate DNA purity by spectrophotometry, measure absorbance from nm to nm in order to detect other possible contaminants present in the DNA solution.

The most common purity calculation is determining the ratio of the absorbance at nm divided by the reading at nm. A reading of 1. However, the best test of DNA quality is functionality in the application of interest e. Strong absorbance around nm can indicate that organic compounds or chaotropic salts are present in the purified DNA.

A ratio of nm to nm can help evaluate the level of salt carryover in the purified DNA. The lower the ratio, the greater the amount of thiocyanate salt is present, for example. A reading at nm will indicate if there is turbidity in the solution, another indication of possible contamination. Therefore, taking a spectrum of readings from nm to nm is most informative.

Agarose gel electrophoresis of the purified DNA eliminates some of the issues associated with absorbance readings. To use this method, a horizontal gel electrophoresis tank with an external power supply, analytical-grade agarose, an appropriate running buffer e.

A sample of the isolated DNA is loaded into a well of the agarose gel and then exposed to an electric field. The negatively charged DNA backbone migrates toward the anode. The percentage of agarose in the gel will determine what size range of DNA will be resolved with the greatest clarity Concentration and yield can be determined after gel electrophoresis is completed by comparing the sample DNA intensity to that of a DNA quantitation standard.

Standards used for quantitation should be labeled as such and be the same size as the sample DNA being analyzed. Because ethidium bromide is a known mutagen, precautions need to be taken for its proper use and disposal DNA-binding dyes compare the unknown sample to a standard curve of DNA, but genomic, fragment and plasmid DNA will each require their own standard curves and cannot be used interchangeably.

If the DNA sample has been diluted, you will need to account for the dilution factor when calculating final concentration. To use this method, a fluorometer to detect the dyes, dilution of the DNA solution and appropriate DNA standards are required. In addition, the usual caveats for handling fluorescent compounds apply—photobleaching and quenching will affect the signal. Choosing which quantitation method to use is based on many factors including access to equipment or reagents, reliability and consistency of the concentration calculations.

Use caution when comparing yields between methods as the level of potential contaminants may cause variable determinations among the different methods.

In this DNA purification guide, we discussed the basic steps of DNA extraction, plasmid preparation and DNA quantitation, and explored the vast portfolio of products that Promega has to offer. This guide is intended to help you understand those basics, navigate issues of scalability, purity, yield and the effects they have on downstream applications, and ultimately assist you in identifying the system that best fits your DNA purification needs.

Need additional assistance? Here at Promega, your success is important to us and we genuinely enjoy the challenge of identifying the right product to address your technical needs. Our Technical Services department is available to help guide you every step of the way, from answering technical questions about your products to providing support for your automated instruments.

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Allow Marketing Cookies. Promega's Cookie Policy We use cookies and similar technologies to make our website work, run analytics, improve our website, and show you personalized content and advertising. Your Account Username Account not found. Email address is unverified. Account is locked. Password Incorrect password. These kits help extract DNA from particular cell types or sample types. However, they can be expensive to use routinely, so many labs have their own methods for DNA extraction.

The cells in a sample are separated from each other, often by a physical means such as grinding or vortexing , and put into a solution containing salt. The positively charged sodium ions in the salt help protect the negatively charged phosphate groups that run along the backbone of the DNA. A detergent is then added. The detergent breaks down the lipids in the cell membrane and nuclei.

DNA is released as these membranes are disrupted. This can be done by a variety of methods. Often a protease protein enzyme is added to degrade DNA-associated proteins and other cellular proteins. In order to study DNA, you first have to get it out of the cell. In eukaryotic cells, such as human and plant cells, DNA is organized as chromosomes in an organelle called the nucleus.

Bacterial cells have no nucleus. Their DNA is organized in rings or circular plasmids, which are in the cytoplasm. Step 1: Lysis In this step, the cell and the nucleus are broken open to release the DNA inside and there are two ways to do this. First, mechanical disruption breaks open the cells.