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- Simple model for point mutation correction uses ssDNA repair oligo and CRISPR-Cas9 RNP
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They then characterized DNA cleavage and repair events, and assessed the cell populations for collateral mutagenesis damage. The team used genotypic and phenotypic readout of a functional and detectable eGFP to assess point mutation correction. The group also examined the targeted region of sorted, clonally expanded, cell populations containing the corrected and uncorrected gene based on eGFP expression to evaluate any collateral mutagenesis left by the action of the gene editing tools. DNA sequence analysis of the eGFP expressing cells showed exact point mutation repair, with no adjacent sequence modification.
However, similar analysis of cell populations containing an uncorrected gene revealed frequent collateral mutagenesis, with deletions and insertions surrounding the target site. In addition, 2 clonal populations that did not express eGFP did in fact show point mutation repair, but also included collateral mutagenesis near the target site. This latter result led the authors to emphasize the importance of analyzing mutagenicity in uncorrected cells.
Technically simple, providing clean electrophoretic results. For single samples or high throughput. Use these programs to calculate T m , identify secondary structure, optimize codon use, select siRNA, and more. Ready for gene construction, genome editing, PCR, or sequencing. Delivered purified and normalized. All rights reserved. Prokaryotic cells are haploid. Some plants are polyploid, for example, modern wheat, which is hexaploid six copies of each chromosome. They are found in a variety of bacterial species, where they behave as additional genetic units inherited and replicated independently of the bacterial chromosome.
However, they rely upon enzymes and proteins provided by the host for their successful transcription and replication. Plasmids often contain genes that code for enzymes that can be advantageous to the host cell in some circumstances. The encoded enzymes may be involved in resistance to, or production of, antibiotics, resistance to toxins found in the environment e. Once purified, plasmid DNA can be used in a wide variety of downstream applications such as sequencing, PCR, expression of proteins, transfection, and gene therapy. DNA can be purified using many different methods and the downstream application determines how pure the DNA should be.
In addition to isolation using home-made methods e. The characteristics of the 3 most common types of DNA extraction kit are shown in the table Characteristics of common DNA extraction kits. Anion-exchange methods yield DNA of a purity and biological activity equivalent to at least two rounds of purification in CsCl gradients, in a fraction of the time.
Purified nucleic acids are of the highest possible quality and are ideal for sensitive downstream biological applications, such as transfection, microinjection, sequencing, and gene therapy research. Silica-membrane technology yields high-purity nucleic acids suitable for most molecular biology and clinical research applications, such as restriction digestion, ligation, labeling, amplification, and radioactive and fluorescent sequencing.
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Magnetic-particle technology yields high-purity nucleic acids suitable for most molecular biology applications used in clinical and veterinary research, such as restriction digestion, ligation, labeling, amplification, and radioactive and fluorescent sequencing. Magnetic-particle technology can often be automated to enable fast and economical nucleic acid purification procedures. Working with DNA: Good laboratory practice.
DNA is a relatively stable molecule. To ensure the integrity of genomic DNA, excessive and rough pipetting and vortexing should be avoided. TE Buffer, pH 7. Spectrophotometric measurement of DNA concentration. For accuracy, absorbance readings at nm should fall between 0. Adapted from reference 1.
Simple model for point mutation correction uses ssDNA repair oligo and CRISPR-Cas9 RNP
Sample storage prior to extraction of genomic DNA. The quality of the starting material affects the quality and yield of the isolated DNA. If samples cannot be processed immediately after harvesting, they should be stored under conditions that preserve DNA integrity. In addition, repeated freezing and thawing of frozen samples should be avoided as this will lead to genomic DNA of reduced size or to reduced yields of pathogen DNA e.
Recommendations for storage of different starting materials are discussed below. An anticoagulant should be added to blood samples that will be stored. Most biological fluids e. Swabs can be stored dry at room temperature. Formalin fixation and paraffin embedding FFPE is another means of sample storage and is particularly relevant for clinical tissue samples. Therefore, the procedures for tissue removal and fixation should be done as quickly as possible. The resulting chemical reaction leads to cross-links between biomolecules, including cross-links between nucleic acids, between proteins, and between nucleic acids and proteins.
For optimal results, neutral-buffered formalin solution should be used instead of unbuffered or acidic formalin solutions. Neutral buffer slows down the degradation of formalin, whose degradation products are believed to contribute to impairing nucleic acid quality. The ratio of formalin to tissue should be at least to ensure optimal fixation.
This is easy to achieve when working with small tissue specimens, such as needle biopsies. However, when dealing with large tissue samples there may be insufficient formalin for fixation. In this case, sections of the tissue should be cut for formalin fixation. Tissues should be fixed for no more than 24 hours to avoid overfixation.
After fixation in formalin, tissue specimens are embedded in paraffin, a process which consists of several steps. The first step is dehydration, where water is replaced by an alcohol, usually ethanol. This is followed by clearing, where the alcohol is replaced by xylene or a xylene substitute, and by impregnation, where xylene is replaced by paraffin. The final step is embedding, where the entire specimen is surrounded with paraffin.
It is important that tissue specimens are fully dehydrated prior to impregnation, as residual water may lead to sample degradation. We recommend always using fresh alcohol and xylene, to avoid any possibility of carryover of water from previous uses. In addition, paraffin containing additives such as beeswax should be avoided, as they may interfere with recovery of biomolecules. Lysed tissue samples can be stored in a suitable lysis buffer for several months at ambient temperature. Animal and human tissues can also be fixed for storage. We recommend using fixatives such as alcohol and formalin; however, long-term storage of tissues in formalin will result in chemical modification of the DNA.
Fixatives that cause cross-linking, such as osmic acid, are not recommended if DNA will be isolated from the tissue. It is also possible to isolate DNA from paraffin-embedded tissue see Other clinical samples. However, some samples e.
New stem cell gene correction process puts time on researchers’ side
Large samples e. If it is not practical to store frozen samples, a number of methods are available for drying plant tissue, for example, silica gel, food dehydrators, or lyophilizers 3. To prevent DNA degradation, material should be completely desiccated in less than 24 hours. Dried samples should be kept in the dark at room temperature under desiccating or hermetic conditions for long-term storage. Depending on how the sample was handled, the DNA in herbarium and forensic samples may be degraded.
Disrupted plant material can be stored in a suitable lysis buffer for several months at ambient temperature. Mycelium should be harvested directly from a culture dish or liquid culture. For liquid cultures, the cells should be pelleted by centrifugation and the supernatant removed before DNA isolation or storage. Sample disruption for extraction of genomic DNA. Complete disruption and lysis of cell walls and plasma membranes of cells and organelles is an absolute requirement for all genomic DNA isolation procedures.
Incomplete disruption results in significantly reduced yields. Disruption generally involves use of a lysis buffer that contains a detergent for breaking down cellular membranes and a protease for digestion of protein cellular components. The choice of protease depends on the lysis buffer used. Some sample types require additional treatment for efficient lysis; this is described in more detail in Special considerations for isolating genomic DNA from different sample sources.
Rotor—stator homogenizers thoroughly disrupt animal and plant tissues in 5—90 seconds depending on the toughness of the sample. The rotor turns at very high speed causing the sample to be disrupted by a combination of turbulence and mechanical shearing. Foaming of the sample should be kept to a minimum by using properly sized vessels, by keeping the tip of the homogenizer submerged, and by holding the immersed tip to one side of the tube.
Rotor—stator homogenizers are available in different sizes and operate with probes of different sizes. Probes with a diameter of 10 mm or above require larger tubes. In disruption using a bead mill, the sample is agitated at high speed in the presence of beads. Disruption occurs by the shearing and crushing action of the beads as they collide with the cells. Disruption efficiency is influenced by:. The optimal beads to use are 0. It is essential that glass beads are pretreated by washing in concentrated nitric acid. Alternatively, use commercially available acid-washed glass beads.
All other disruption parameters must be determined empirically for each application. For disruption using a mortar and pestle, freeze the sample immediately in liquid nitrogen and grind to a fine powder under liquid nitrogen. Transfer the suspension tissue powder and liquid nitrogen into a liquid-nitrogen—cooled, appropriately sized tube and allow the liquid nitrogen to evaporate without allowing the sample to thaw. Add lysis buffer and continue as quickly as possible with the isolation procedure. Some sample sources contain substances that can cause problems in DNA isolation and analysis.
Special considerations are required when working with these sample sources. In this section, considerations for working with a number of different sources are discussed. Human blood samples are routinely collected for clinical analysis. Blood contains a number of enzyme inhibitors that can interfere with downstream DNA analysis.
In addition, common anticoagulants such as heparin and EDTA can interfere with downstream assays. DNA isolation from blood requires a method to provide high-quality DNA without contaminants or enzyme inhibitors. In animals, erythrocytes red blood cells from birds, fish, and frogs contain nuclei and hence genomic DNA, while those from mammals do not.
Since healthy mammalian blood contains approximately times more erythrocytes than nuclei-containing leukocytes white blood cells, comprising lymphocytes, monocytes, and granulocytes removing the erythrocytes prior to DNA isolation can give higher DNA yields. This can be accomplished by several methods. One is selective lysis of erythrocytes, which are more susceptible than leukocytes to hypotonic shock and burst rapidly in the presence of a hypotonic buffer.
Alternatively, Ficoll density-gradient centrifugation can be performed to recover mononuclear cells lymphocytes and monocytes and remove erythrocytes. This technique also removes granulocytes. A third method is to prepare a leukocyte-enriched fraction of whole blood, called buffy coat, by centrifuging whole blood at x g for 10 minutes at room temperature. After centrifugation, three different fractions are distinguishable: the upper clear layer is plasma; the intermediate layer is buffy coat; and the bottom layer contains concentrated erythrocytes.
Blood samples, including those treated to remove erythrocytes, can be efficiently lysed using lysis buffer and protease or proteinase K. Most biological fluids can be treated in the same way as blood samples for isolation of DNA. Isolation of DNA from stool samples is more difficult, as stool typically contains many compounds that can degrade DNA and inhibit downstream enzymatic reactions.
Simple model for point mutation correction uses ssDNA repair oligo and CRISPR-Cas9 RNP
Animal cell cultures and most animal tissues can be efficiently lysed using lysis buffer and protease or proteinase K. Fresh or frozen samples should be cut into small pieces to aid lysis. Mechanical disruption using a homogenizer or mortar and pestle prior to lysis can reduce lysis time. Skeletal muscle, heart, and skin tissue have an abundance of contractile proteins, connective tissue, and collagen, and care should be taken to ensure complete digestion with protease or proteinase K.
For fixed tissues, the fixative should be removed prior to lysis. Formalin can be removed by washing the tissue in phosphate-buffered saline PBS. Paraffin should be similarly removed from paraffin-embedded tissues by extraction with xylene followed by washing with ethanol. Yeast cell cultures must first be treated with lyticase or zymolase to digest the cell wall. The resulting spheroplasts are collected by centrifugation and then lysed using lysis buffer and proteinase K or protease. Many bacterial cell cultures can be efficiently lysed using lysis buffer and protease or proteinase K.
Some bacteria, particularly Gram-positive bacteria, require pre-incubation with specific enzymes e. Bacterial DNA can also be isolated from a wide variety of clinical samples. Bacterial cells should be pelleted from biological fluids, and the DNA isolated as for bacterial cell cultures. Swab samples should be pretreated with fungicide before centrifugation of bacterial cells. In clinical applications, viral DNA is often although not always isolated from cell-free body fluids, where their titer can be very low. Virus particles may need to be concentrated before DNA isolation by ultracentrifugation, ultrafiltration, or precipitation.
Bacteriophage, such as M13 and lambda, are isolated from infected bacterial cultures. The bacterial cells must be removed from the culture by centrifugation prior to isolation of viral DNA. Isolation of DNA from plant material presents special challenges, and commonly used techniques often require adaptation before they can be used with plant samples. Several plant metabolites have chemical properties similar to those of nucleic acids, and are difficult to remove from DNA preparations.
Co-purified metabolites and contaminants introduced by the purification procedure, such as salts or phenol, can inhibit enzymatic reactions or cause variations in UV spectrophotometric measurements and gel migration. DNA isolation is often improved by using plants grown under conditions that do not induce high levels of plant metabolites. Because of the great variation among plants, it is difficult to make general statements about growth conditions to use. However, as a general guideline, it is recommended to use healthy, young tissues when possible.
DNA yields from young tissues are often higher than from old tissue because young tissue generally contains more cells than the same amount of older tissue. Young tissue of the same weight also contains fewer metabolites. Working with DNA: Good microbiological practice. Good microbiological technique will always ensure the best yield and quality of plasmid DNA. To prepare the perfect bacterial culture for your plasmid prep, follow the steps below. The growth curve of an E. The first, lag phase , occurs directly after dilution of the starter culture into fresh medium.
During this phase, cell division is slow as the bacteria adapt to the fresh medium. The bacteria then start to divide more rapidly and the culture enters logarithmic log phase 4—5 hours after dilution , during which the number of cells increases exponentially.
Eventually the culture enters the phase of decline as cells start to lyse, the number of viable bacteria falls, and DNA becomes partly degraded. There are different methods for storing E. Glycerol stocks and stab cultures enable long-term storage of bacteria, while agar plates can be used for short-term storage. Preparation instructions and useful tips for each of these methods are given below.
Stab cultures are used to transport or send bacterial strains to other labs. Bacteria should always be streaked onto plates containing the appropriate antibiotic to ensure that selective markers are not lost. The figure, Essential steps for storage and handling of E. Bacterial stocks should always be streaked onto selective plates prior to use, to check that they give rise to healthy colonies carrying the appropriate antibiotic resistance.
Stocks can potentially contain mutants arising from the cultures used to prepare them, or can deteriorate during storage. Inoculate liquid cultures from a healthy, well-isolated colony, picked from a freshly streaked selective plate. This will ensure that cells growing in the culture are all descended from a single founder cell, and have the same genetic makeup.
Plasmids vary widely in their copy number see table Origin of replication and copy numbers of various plasmids and cosmids , depending on the origin of replication they contain pMB1 or pSC for example which determines whether they are under relaxed or stringent control; as well as the size of the plasmid and its associated insert. Some plasmids, such as the pUC series and derivatives, have mutations which allow them to reach very high copy numbers within the bacterial cell.
Plasmids based on pBR and many cosmids are generally maintained at lower copy numbers. Very large plasmids are often maintained at very low copy numbers per cell. The high-copy plasmids listed here contain mutated versions of this origin. Bacterial cultivation media and antibiotics. Liquid cultures of E. Please note, however, that a number of different LB broths, with different compositions, are commonly used. Different formulations contain different concentrations of NaCl and give rise to varied yields of plasmid DNA.
For preparation of 1 liter of LB medium, add 10 g NaCl, 10 g tryptone, and 5 g yeast extract to ml distilled or deionized water, and shake or stir until dissolved. Adjust the pH to 7. Adjust the volume of the solution to 1 liter with distilled or deionized water. Decant into smaller aliquots and sterilize by autoclaving see Sterilizing media. Tip : It is advisable to autoclave liquid medium in several small bottles rather than in one large vessel to avoid possible contamination of an entire batch. After autoclaving, do not use medium for 24 hours to ensure that it is properly sterilized and free of contaminating microorganisms.
Sterilize liquid or solid media by autoclaving, using a pressure and time period suitable for the type of medium, bottle size, and autoclave type. Tip : Antibiotics and nutrients such as amino acids are inactivated by the high temperatures of an autoclave. They should be sterilized by filtration through a filter unit with a pore size of 0.
Preparation : Prepare LB medium according to the composition given in Liquid media. Just before autoclaving, add 15 g agar per liter and mix. After autoclaving, swirl the medium gently to distribute the melted agar evenly throughout the solution. Take care that the hot liquid does not boil over when swirled. Mix thoroughly to obtain an even concentration throughout the medium before pouring.
Tip : Pour plates in a laminar-flow hood or, if no hood is available, on a cleaned bench surface next to a Bunsen. After pouring plates, any air bubbles may be removed by passing the flame of a Bunsen burner briefly over the surface. Do not linger with the flame as this may destroy antibiotics in sections of the plates. Dry plates either directly after solidification or just before use by removing the lids and standing the plates in a laminar-flow hood for 1 hour.
Do not store for longer than 3 months as antibiotics may degrade. Bacterial strains carrying plasmids or genes with antibiotic selection markers should always be cultured in liquid or on solid medium containing the selective agent. Lack of antibiotic selection can lead to loss of the plasmid carrying the genetic marker and potentially to selection of faster-growing mutants! Recommended stock and working concentrations for commonly used antibiotics are shown in the table Concentrations of commonly used antibiotics. Lysis of bacterial cells for plasmid purification.
Effective lysis of bacterial cells is a key step in plasmid isolation as DNA yield and quality depend on the quality of cell lysate used for the purification. Alkaline lysis is one of the most commonly used methods for lysing bacterial cells prior to plasmid purification 4, 5. Production of alkaline lysates involves four basic steps see figure The principle of alkaline lysis.
A number of other methods have been described for lysing bacterial cells 1, 6. Some of these methods were developed for other applications and may not be suitable for plasmid DNA preparation. Isolation of plasmid DNA from bacteria other than E. Several techniques exist to prepare competent cells and one such technique for preparing competent E. Transformation is the process in which plasmid DNA is introduced into a bacterial host cell. Several methods exist for transformation of bacterial cells, one of which is given below. Transform competent cells with 1 ng of a control plasmid containing an antibiotic resistance gene.
Plate onto LB-agar plates containing the relevant antibiotic s. Compare the number of colonies obtained with the control plasmid to the number obtained with the plasmid of interest to compare transformation efficiency. An absence of colonies on the plates indicates that the antibiotic is active. Alcohol precipitation is commonly used for concentrating, desalting, and recovering nucleic acids. Precipitation is mediated by high concentrations of salt and the addition of either isopropanol or ethanol.
Since less alcohol is required for isopropanol precipitation, this is the preferred method for precipitating DNA from large volumes. In addition, isopropanol precipitation can be performed at room temperature, which minimizes co precipitation of salt that interferes with downstream applications. Avoid repeated freeze-thawing as this will lead to precipitates.
Diluted solutions of nucleic acids e. This avoids adsorption of nucleic acids to the tube walls, which would reduce the concentration of nucleic acids in solution. Endotoxins, also known as lipopolysaccharides or LPS, are cell membrane components of Gram-negative bacteria e. A single E. Therefore, each endotoxin molecule possesses hydrophobic, hydrophilic, and charged regions giving it unique features with respect to possible interactions with other molecules.
Bacteria shed small amounts of endotoxins into their surroundings while they are actively growing and large amounts when they die. During lysis of bacterial cells for plasmid preparations, endotoxin molecules are released from the outer membrane into the lysate. Endotoxins significantly reduce transfection efficiencies in endotoxin-sensitive cell lines. Overall, endotoxins represent a non-controllable variable in transfection experiment setup.
They are invisible on agarose gels and impossible to detect by optical density and influence the outcome and reproducibility of results and making them difficult to compare and interpret. The chemical structure and properties of endotoxin molecules and their tendency to form micellar structures lead to copurification of endotoxins with plasmid DNA. For example, in CsCl ultracentrifugation, the CsCl-banded DNA is easily contaminated with endotoxin molecules, which have a similar density in CsCl to plasmid—ethidium bromide complexes. On size-exclusion resins, the large size of the micellar form of endotoxin causes the molecule to behave like a large DNA molecule; and in anion-exchange chromatography, the negative charges present on the endotoxin molecule can interact with anion-exchange resins, thus leading to copurification of endotoxins with the plasmid DNA.
However, the level of endotoxin contamination found in plasmid DNA is dependent on the purification method used.
Historically, endotoxins were measured in a clotting reaction between the endotoxin and a clottable protein in the amoebocytes of Limulus polyphemus , the horseshoe crab. Today much more sensitive photometric tests e. LPS contamination is usually expressed in endotoxin units EU. Endotoxins strongly influence transfection of DNA into primary cells and sensitive cultured cells, and increased endotoxin levels lead to sharply reduced transfection efficiencies. Furthermore, it is extremely important to use endotoxin-free plasmid DNA for gene therapy applications, since endotoxins cause fever, endotoxic shock syndrome, and activation of the complement cascade in animals and humans.
Endotoxins also interfere with in vitro transfection into immune cells such as macrophages and B cells by causing nonspecific activation of immune responses. These responses include the induced synthesis of immune mediators such as IL-1 and prostaglandin. It is important to make sure that plasticware, media, sera, and plasmid DNA are free of LPS contamination to avoid misinterpretation of experimental results. To avoid recontamination of plasmid DNA after initial endotoxin removal, we recommend using only new plasticware which is certified to be pyrogen- or endotoxin-free.
Endotoxin-free or pyrogen-free plasticware can be obtained from many different suppliers. Endotoxins adhere strongly to glassware and are difficult to remove completely during washing. Standard laboratory autoclaving procedures have little or no effect on endotoxin levels.
Moreover, if the autoclave has previously been used for bacteria, the glassware will become extensively contaminated with endotoxin molecules. It is also important not to recontaminate the purified endotoxin-free DNA by using reagents that are not endotoxin-free. Reliable measurement of DNA concentration is important for many applications in molecular biology. Spectrophotometry and fluorometry are commonly used to measure both genomic and plasmid DNA concentration.
Spectrophotometry can be used to measure microgram quantities of pure DNA samples i. Fluorometry is more sensitive, allowing measurement of nanogram quantities of DNA, and furthermore, the use of Hoechst dye allows specific analysis of DNA. DNA concentration can be determined by measuring the absorbance at nm A in a spectrophotometer using a quartz cuvette. For greatest accuracy, readings should be between 0. This relation is valid only for measurements made at neutral pH, therefore, samples should be diluted in a low-salt buffer with neutral pH e.
An example of the calculation involved in nucleic acid quantification when using a spectrophotometer see Spectrophotometric measurement of DNA concentration. When working with small amounts of DNA, such as purified PCR products or DNA fragments extracted from agarose gels, quantification via agarose gel analysis may be more effective see Agarose gel.
Tip : If you use more than one cuvette to measure multiple samples, the cuvettes must be matched. Tip : Phenol has an absorbance maximum of — nm, which is close to that of DNA. Phenol contamination mimics both higher yields and higher purity, because of an upward shift in the A value. Absorption of nucleic acids depends on the solvent used to dissolve the nucleic acid 7. A values are reproducible when using low-salt buffer, but not when using water.
This is most likely due to differences in the pH of the water caused by the solvation of CO 2 from air. RNA contamination can sometimes be detected by agarose gel analysis with routine ethidium bromide staining, although not quantified effectively.
here Prior to use, ensure that the RNase A solution has been heat-treated to destroy any contaminating DNase activity. Alternatively, use DNase-free RNase purchased from a reliable supplier. RNA contamination of plasmid DNA can be a concern depending on the method used for plasmid preparation.
Be sure to zero the spectrophotometer with the appropriate buffer. Scanning the absorbance from — nm will show whether there are contaminants affecting absorbance at nm. Absorbance scans should show a peak at nm and an overall smooth shape. Fluorometry allows specific and sensitive measurement of DNA concentration by use of a fluorescent dye; with common dyes including Hoechst dyes and PicoGreen.
It shows increased emission at nm when bound to DNA. DNA standards and samples are mixed with Hoechst and measured in glass or acrylic cuvettes using a scanning fluorescence spectrophotometer or a dedicated filter fluorometer set at an excitation wavelength of nm and an emission wavelength of nm. The sample measurements are then compared to the standards to determine DNA concentration. As little as 20 ng DNA can be detected by agarose gel electrophoresis with ethidium bromide staining.
The amount of sample DNA loaded can be estimated by comparison of the band intensity with the standards either visually see figure Agarose gel analysis of plasmid DNA or using a scanner or imaging system. Be sure to use standards of roughly the same size as the fragment of interest to ensure reliable estimation of the DNA quantity, since large fragments interchelate more dye than small fragments and give a greater band intensity.
More precise agarose gel quantification can be achieved by densitometric measurement of band intensity and comparison with a standard curve generated using DNA of a known concentration. In most experiments the effective range for comparative densitometric quantification is between 20 and ng. Tip : The amount of DNA used for densitometric quantification should fall within the linear range of the standard curve. See DNA analysis using analytical gels , for further information on agarose gel electrophoresis.
Restriction endonuclease digestion of DNA. Many applications require conversion of genomic DNA into conveniently sized fragments by restriction endonuclease digestion. This yields DNA fragments of a convenient size for downstream manipulations. Restriction endonucleases are bacterial enzymes that bind and cleave DNA at specific target sequences. Type II restriction enzymes are the most widely used in molecular biology applications. They bind DNA at a specific recognition site, consisting of a short palindromic sequence, and cleave within this site, e. Isoschizomers are different enzymes that share the same specificity, and in some cases, the same cleavage pattern.
Tip : Isoschizomers may have slightly different properties that can be very useful. Sau 3A can therefore be used instead of Mbo I where necessary. Restriction enzymes with shorter recognition sequences cut more frequently than those with longer recognition sequences. For example, a 4 base pair bp cutter will cleave, on average, every 4 4 bases, while a 6 bp cutter cleaves every 4 6 bases. Many organisms have enzymes called methylases that methylate DNA at specific sequences.
Not all restriction enzymes can cleave their recognition site when it is methylated. Therefore the choice of restriction enzyme is affected by its sensitivity to methylation. In addition, methylation patterns differ in different species, also affecting the choice of restriction enzyme. Tip : Methylation patterns differ between bacteria and eukaryotes, so restriction patterns of cloned and uncloned DNA may differ.
Tip : Methylation patterns also differ between different eukaryotes see bullets above , affecting the choice of restriction enzyme for construction genomic DNA libraries. Some restriction enzymes cut in the middle of their recognition site, creating blunt-ended DNA fragments.
Some enzymes create 5' overhangs and others create 3' overhangs. The type of digestion affects the ease of downstream cloning:. If a DNA fragment is to be cut with more than one enzyme, both enzymes can be added to the reaction simultaneously provided that they are both active in the same buffer and at the same temperature. If the enzymes do not have compatible reaction conditions, it is necessary to carry out one digestion, purify the reaction products, and then perform the second digestion. The amount of DNA digested depends on the downstream application and the genome size of the organism being analyzed.
For mapping of cloned DNA, 0. Tip : DNA should be free from contaminants such as phenol, chloroform, ethanol, detergents, or salt, as these may interfere with restriction endonuclease activity. Most researchers add a fold excess of enzyme to their reactions in order to ensure complete cleavage.
The products of the ligation mixture are introduced into competent E. Appropriate control ligations should also be performed see Preparation of competent E. Removal of 5' phosphates from linearized vector DNA can help prevent vector self-ligation and improve ligation efficiency. Controls are essential if things go wrong.
For example, colonies on plates that receive mock-transformed bacteria may indicate that the medium lacks the correct antibiotic. An absence of colonies on plates receiving bacteria transformed with plasmids under construction can only be interpreted if a positive control using a standard DNA has been included. See Bacterial cultivation media and antibiotics for further information on transformation controls.
Gels allow separation and identification of nucleic acids based on charge migration. Migration of nucleic acid molecules in an electric field is determined by size and conformation, allowing nucleic fragments of different sizes to be separated. However, the relationship between the fragment size and rate of migration is non-linear, since larger fragments have greater frictional drag and are less efficient at migrating through the polymer. Agarose gel analysis is the most commonly used method for analyzing DNA fragments between 0.
This section provides useful hints for effective gel analysis of DNA. The concentration of agarose used for the gel depends primarily on the size of the DNA fragments to be analyzed. Low agarose concentrations are used to separate large DNA fragments, while high agarose concentrations allow resolution of small DNA fragments see table Concentration of agarose used for separating fragments of different sizes.
Most gels are run using standard agarose, although some special types of agarose are available for particular applications. For example, low-melt agarose allows in situ enzymatic reactions and can therefore be used for preparative gels. Genomic DNA can be isolated directly from cells immobilized in low-melt agarose gels see reference 6 for more information. Tip : Use ultrapure-quality agarose since impurities such as polysaccharides, salts, and proteins can affect the migration of DNA.
Agarose quality is particularly important when running high-percentage agarose gels. For example, low-melt agarose allows in situ enzymatic reactions and can be used for preparative gels. Although more frequently used, TAE has a lower buffering capacity than TBE and is more easily exhausted during extended electrophoresis. The drawback of TBE is that the borate ions in the buffer form complexes with the cis-diol groups of sugar monomers and polymers, making it difficult to extract DNA fragments from TBE gels using traditional methods.
TBE 0. Agarose gel electrophoresis allows analysis of DNA fragments between 0. The amount of genomic DNA loaded onto a gel depends on the application, but in general, loading of too much DNA should be avoided as this will result in smearing of the DNA bands on the gel. Gel loading buffer see table Gel loading buffer must be added to the samples before loading and serves three main purposes:.
Molecular-weight markers should always be included on a gel to enable analysis of DNA fragment sizes in the samples. See table Commonly used DNA markers in agarose gel electrophoresis for commonly used markers. To allow visualization of the DNA samples, agarose gels are stained with an appropriate dye. The most commonly used dye is the intercalating fluorescent dye ethidium bromide, which can be added either before or after the electrophoresis see table Comparison of ethidium bromide staining methods. Addition of ethidium bromide prior to electrophoresis — add ethidium bromide at a concentration of 0.
Mix the agarose—ethidium bromide solution well to avoid localized staining. Addition of ethidium bromide after electrophoresis — soak the gel in a 0. Tip : Rinse the gel with buffer or water before examining it to remove excess ethidium bromide. Tip : Staining buffer can be saved and re-used. Note : Ethidium bromide is a powerful mutagen and is very toxic. Wear gloves and take appropriate safety precautions when handling. Use of nitrile gloves is recommended as latex gloves may not provide full protection.
After use, ethidium bromide solutions should be decontaminated as described in commonly used manuals 1, 6. Ethidium bromide—DNA complexes display increased fluorescence compared to the dye in solution. This means that illumination of a stained gel under UV light — nm allows bands of DNA to be visualized against a background of unbound dye.
The gel image can be recorded by taking a Polaroid photograph or using a gel documentation system. Tip : UV light can damage the eyes and skin. Always wear suitable eye and face protection when working with a UV light source. Southern blotting is a widely used technique that allows analysis of specific DNA sequences. DNA is usually first converted into conveniently sized fragments by restriction digestion. The DNA is next run through an agarose gel 6.