Laboratory Methods in Enzymology: DNA

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Using western blotting techniques allows not only detection but also quantitative analysis. Analogous methods to western blotting can be used to directly stain specific proteins in live cells or tissue sections. The eastern blotting technique is used to detect post-translational modification of proteins.

Proteins blotted on to the PVDF or nitrocellulose membrane are probed for modifications using specific substrates. A DNA microarray is a collection of spots attached to a solid support such as a microscope slide where each spot contains one or more single-stranded DNA oligonucleotide fragments.

A History of Nucleic Acid Preparation Tools

Arrays make it possible to put down large quantities of very small micrometre diameter spots on a single slide. A variation of this technique allows the gene expression of an organism at a particular stage in development to be qualified expression profiling. This cDNA is then hybridized to the fragments on the array and visualization of the hybridization can be done.

Since multiple arrays can be made with exactly the same position of fragments they are particularly useful for comparing the gene expression of two different tissues, such as a healthy and cancerous tissue. Also, one can measure what genes are expressed and how that expression changes with time or with other factors. There can be anywhere from spots to more than 10, on a given array. Arrays can also be made with molecules other than DNA. Allele-specific oligonucleotide ASO is a technique that allows detection of single base mutations without the need for PCR or gel electrophoresis.

Short 20—25 nucleotides in length , labeled probes are exposed to the non-fragmented target DNA, hybridization occurs with high specificity due to the short length of the probes and even a single base change will hinder hybridization. The target DNA is then washed and the labeled probes that didn't hybridize are removed. The target DNA is then analyzed for the presence of the probe via radioactivity or fluorescence. In this experiment, as in most molecular biology techniques, a control must be used to ensure successful experimentation.

In molecular biology, procedures and technologies are continually being developed and older technologies abandoned. For example, before the advent of DNA gel electrophoresis agarose or polyacrylamide , the size of DNA molecules was typically determined by rate sedimentation in sucrose gradients , a slow and labor-intensive technique requiring expensive instrumentation; prior to sucrose gradients, viscometry was used.

Aside from their historical interest, it is often worth knowing about older technology, as it is occasionally useful to solve another new problem for which the newer technique is inappropriate. While molecular biology was established in the s, the term was coined by Warren Weaver in Weaver was the director of Natural Sciences for the Rockefeller Foundation at the time and believed that biology was about to undergo a period of significant change given recent advances in fields such as X-ray crystallography. Clinical research and medical therapies arising from molecular biology are partly covered under gene therapy.

The use of molecular biology or molecular cell biology approaches in medicine is now called molecular medicine. Molecular biology also plays important role in understanding formations, actions, and regulations of various parts of cells which can be used to efficiently target new drugs , diagnose disease, and understand the physiology of the cell. From Wikipedia, the free encyclopedia. For the scientific journal, see Biochemical Genetics. Branch of biology dealing with biological activity's molecular basis. Index Outline. For more extensive list on protein methods, see protein methods.

For more extensive list on nucleic acid methods, see nucleic acid methods. Main article: Molecular cloning. Main article: Polymerase chain reaction. Main article: Gel electrophoresis. Main article: Southern blot. Main article: Northern blot. Main article: Western blot. Main article: Eastern blot. Main article: DNA microarray. Play media. Main article: Allele-specific oligonucleotide. Main article: History of molecular biology. Central dogma of molecular biology Genetic code Genome Molecular biology institutes Molecular engineering Molecular microbiology Molecular modeling Protein interaction prediction Protein structure prediction Proteome Cell biology.

Molecular Biology of the Cell, Sixth Edition. Garland Science. Bibcode : Natur. Molecular cell biology 4th ed. New York: Scientific American Books. Biochemistry 5th ed. W H Freeman. Genetics Home Reference. Retrieved 31 December Molecular Imaging: Fundamentals and Applications. Retrieved Molecular cloning. Methods in Enzymology. Textbook of Pharmaceutical Biotechnology. Expression Cloning. Journal of Visualized Experiments Bibcode : PNAS Southern blotting.

Current Protocols in Immunology. Chapter Unit Nielsen, Henrik ed. RNA methods and protocols. Methods in Molecular Biology. New York: Humana Press. Northern blot. North American Journal of Medical Sciences. Hal 1 April H 1 June Parasite Immunology. Frederick M. Ausubel; et al. Current Protocols in Molecular Biology.

Chapter 22, Unit— American Journal of Obstetrics and Gynecology. Molecular genetic pathology. Totowa, NJ: Humana. Molecular Pathology in Clinical Practice. 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 Digestion of DNA

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.


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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. Southern blotting named after its inventor, E.

Southern refers to the transfer of the DNA to a nylon or nitrocellulose membrane by capillary transfer. The DNA of interest can be identified by hybridization to radioactive or chemiluminescent probes and visualized by autoradiography or staining. Many variations on the Southern blotting procedure exist. A standard protocol is described here together with recipes for buffers and solutions.

DNA fragments longer than 10 kb do not transfer to blotting membranes efficiently. In order to facilitate their transfer, these fragments are reduced in size, either by acid depurination or by UV irradiation. Acid depurination — immediately after gel electrophoresis, place the gel in a solution of 0. Agitate gently for 10 minutes. During this period the color of the bromophenol blue in the samples will change from blue to yellow, indicating that the gel has been completely saturated with the acid.

Rinse the gel briefly in distilled water. Tip : The depurination step should not last too long, since very short fragments attach less firmly to the membrane. Depurination is therefore recommended only when fragments larger than 10 kb are to be transferred. UV irradiation — expose the gel to UV light at a wavelength of nm from a source operating at 30 W, for 30—60 seconds. Double-stranded DNA must be denatured in order to create suitable hybridization targets. Completely cover the gel with denaturation buffer see table Denaturation buffer and incubate for 30 minutes with gentle shaking.

If acid depurination was used to denature the DNA, the bromophenol blue will return to its original color during this incubation. Remove the denaturation buffer and completely cover the gel in neutralization buffer see table Neutralization buffer. Incubate for 30 minutes with gentle shaking. Adjust to pH 7.

Cleanup of DNA is often a prerequisite for efficient downstream applications such as cloning, sequencing, microarray analysis, or amplification. The use of a dedicated kit for this application may be necessary, since kits can vary depending on the type of reaction and DNA fragment size e. In next-generation sequencing NGS library preparation, often, much larger primers — almost in the range of a PCR amplicon — must be removed, and PCR cleanup may not be sufficient.

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Evaluation of Methods for the Extraction and Purification of DNA from the Human Microbiome

Log Out. Print Bookmark Share. This section describes considerations for isolation and quantification of both genomic DNA from different sample sources and plasmid DNA. It also deals with common plasmid DNA procedures, including how to make and transform competent cells, how to culture and handle plasmid-containing cells, and commonly used techniques for analysis of genomic DNA.

What is DNA? Strong absorbance at nm and nm may indicate the presence of contaminating phenol. Absorbance at nm suggests contamination by particulates in the solution or dirty cuvettes. Blood An anticoagulant should be added to blood samples that will be stored. Other clinical samples Most biological fluids e. Fungal material Mycelium should be harvested directly from a culture dish or liquid culture. Back to top 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.

Disruption methods Lysis buffer 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. Disruption using rotor—stator homogenizers Rotor—stator homogenizers thoroughly disrupt animal and plant tissues in 5—90 seconds depending on the toughness of the sample. Disruption using bead mills In disruption using a bead mill, the sample is agitated at high speed in the presence of beads.

Disruption using a mortar and pestle For disruption using a mortar and pestle, freeze the sample immediately in liquid nitrogen and grind to a fine powder under liquid nitrogen. Special considerations for isolating genomic DNA from different sample sources Some sample sources contain substances that can cause problems in DNA isolation and analysis.

Blood Human blood samples are routinely collected for clinical analysis. Other clinical samples Most biological fluids can be treated in the same way as blood samples for isolation of DNA. Animal tissues and cell culture Animal cell cultures and most animal tissues can be efficiently lysed using lysis buffer and protease or proteinase K. Yeast cell cultures Yeast cell cultures must first be treated with lyticase or zymolase to digest the cell wall. Bacterial DNA Many bacterial cell cultures can be efficiently lysed using lysis buffer and protease or proteinase K.

DNA viruses In clinical applications, viral DNA is often although not always isolated from cell-free body fluids, where their titer can be very low. Plants Isolation of DNA from plant material presents special challenges, and commonly used techniques often require adaptation before they can be used with plant samples. Prepare a starter culture by inoculating a single colony from a freshly streaked selective plate into 2—10 ml LB Luria-Bertani medium containing the appropriate antibiotic. Tip : Do not inoculate directly from glycerol stocks, agar stabs, or plates that have been stored for a long time, as this may lead to loss or mutation of the plasmid.

Tip : It is often convenient to grow the starter culture during the day so that the larger culture can be grown overnight for harvesting the following morning. Use a flask of at least 4 times the volume of culture to ensure sufficient aeration. Do not use a larger culture volume than recommended in the protocol, as this will result in inefficient lysis and reduce the quality of the preparation. Harvest the bacterial culture 12—16 hours after inoculation. This corresponds to the transition from logarithmic into stationary growth phase see figure Growth curve of E. Harvesting too early may result in lower than expected yields of plasmid DNA due to a lower cell density.

Harvesting too late may result in low plasmid quality and yield due to DNA degradation from over-aging of the culture. Tip : Growth of cultures is dependent on factors such as host strain, plasmid insert and copy number, and culture medium. To determine the optimal harvesting time for a particular system, monitor the cell density and the growth of the culture by measuring the OD see next section.

Remove all traces of supernatant by inverting the open centrifuge tube until all of the medium has been drained. The cells are now ready for the lysis procedure, as indicated in the appropriate plasmid purification protocol. The procedure may be stopped at this point and continued later by freezing the cell pellets obtained by centrifugation. The E. Back to top Storage of E. Glycerol stocks E. Prepare glycerol stocks of bacteria as follows: Add 0.

Introduction

Tip : Vials of sterilized glycerol can be prepared in batches and stored at room temperature until required. Add 0. Vortex the vial vigorously to ensure even mixing of the bacterial culture and the glycerol. Tip : Avoid repeated thawing and re-freezing of glycerol stocks as this can reduce the viability of the bacteria. Tip : For precious strains, storage of 2 stock vials is recommended.

DNA Amplification Techniques

Tip : When recovering a stored strain, it is advisable to check the antibiotic markers by streaking the strain onto a selective plate. Stab cultures E. While the agar is still liquid, add 1 ml agar to a 2 ml screw-cap vial under sterile conditions, then leave to solidify. Vials of agar can be prepared in batches and stored at room temperature until required. Using a sterile straight wire, pick a single colony from a freshly streaked plate and stab it deep down into the soft agar several times see figure Inoculating a stab culture.

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When recovering a stored strain, it is advisable to check the antibiotic markers by streaking the strain onto a selective plate. To obtain well-isolated colonies, streak an agar plate as follows: Flame a wire loop, and cool on a spare sterile agar plate. Using the wire loop, streak an inoculum of bacteria from a glycerol stock, stab culture, or single colony on another plate across one corner of a fresh agar plate, as shown in the figure Streaking bacteria on agar plates.

Flame and cool the wire loop again. Pass it through the first streak and then streak again across a fresh corner of the plate. Repeat again to form a pattern. Generating liquid cultures from bacterial stocks The figure, Essential steps for storage and handling of E. Back to top Plasmid specifications 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.

Back to top Bacterial cultivation media and antibiotics Liquid media Liquid cultures of E. Sterilizing media Sterilize liquid or solid media by autoclaving, using a pressure and time period suitable for the type of medium, bottle size, and autoclave type. Solid media E. Antibiotics Bacterial strains carrying plasmids or genes with antibiotic selection markers should always be cultured in liquid or on solid medium containing the selective agent. Alkaline lysis Alkaline lysis is one of the most commonly used methods for lysing bacterial cells prior to plasmid purification 4, 5.

Tip : Ensure that bacteria are resuspended completely leaving no cell clumps in order to maximize the number of cells exposed to the lysis reagents. Tip : For large-scale purification of low-copy plasmids, for which larger cultures volumes are used, it may be beneficial to increase the lysis buffer volumes in order to increase the efficiency of alkaline lysis and thereby the DNA yield.

Sodium dodecyl sulfate SDS solubilizes the phospholipid and protein components of the cell membrane, leading to lysis and release of the cell contents. This results in insufficient cell lysis and it is recommended to double the amount of lysis and neutralization buffers used. Tip : Avoid vigorous stirring or vortexing of the lysate as this can shear the bacterial chromosome, which will then copurify with the plasmid DNA. The solution should be mixed gently but thoroughly by inverting the lysis vessel 4—6 times. Tip : Do not allow the lysis to proceed for longer than 5 minutes.

This is optimal for release of the plasmid DNA, while avoiding irreversible plasmid denaturation. Neutralize the lysate by adding acidic potassium acetate. Note: The high salt concentration causes potassium dodecyl sulfate KDS to precipitate, and denatured proteins, chromosomal DNA, and cellular debris are coprecipitated in insoluble salt-detergent complexes. Plasmid DNA, being circular and covalently closed, renatures correctly and remains in solution.

Tip : Precipitation can be enhanced by using chilled neutralization buffer and incubating on ice. Clear the lysate by either centrifugation or filtration, to precipitate the debris. Note : Purification of plasmid DNA from cleared bacterial lysates was traditionally performed using cesium chloride CsCl ultracentrifugation. Today, a variety of commercially available plasmid purification kits offer easy procedures for different throughput requirements and applications. Other lysis methods A number of other methods have been described for lysing bacterial cells 1, 6. Lysis with detergent : Bacterial cells are lysed by treatment with and ionic detergent e.

Mechanical lysis : Bacterial cells are lysed by mechanical disruption e. Enzymatic digestion : Some lysis methods include treatment of bacteria with enzymes such as lysozyme which assist in weakening cell walls. Lysis of bacteria other than E. Note : Cells prepared using this protocol are not suitable for electroporation. Materials required E. Remove a trace of E. Pick a single colony and inoculate 10 ml LB medium containing relevant antibiotic s. Cool the culture on ice for 5 min, and transfer the culture to a sterile, round-bottom centrifuge tube.

Discard the supernatant carefully. Always keep the cells on ice. Resuspend the cells carefully in 4 ml ice-cold TFB2 buffer. Back to top Transformation of competent E. Competent E. Thaw an aliquot of frozen competent E. Tip : Shaking increases transformation efficiency.

Positive control to check transformation efficiency Transform competent cells with 1 ng of a control plasmid containing an antibiotic resistance gene. Back to top Isopropanol precipitation of DNA Alcohol precipitation is commonly used for concentrating, desalting, and recovering nucleic acids. Adjust the salt concentration if necessary, for example, with sodium acetate 0. Tip : Use all solutions at room temperature to minimize co-precipitation of salt. Tip : Do not use polycarbonate tubes for precipitation as polycarbonate is not resistant to isopropanol. When precipitating from small volumes, centrifugation may be carried out at room temperature.

The spooled DNA should be transferred immediately to a microfuge tube containing an appropriate buffer and redissolved see step 9.

Carefully decant the supernatant without disturbing the pellet. Tip : Marking the outside of the tube before centrifugation allows the pellet to be more easily located. Pellets from isopropanol precipitation have a glassy appearance and may be more difficult to see than the fluffy salt-containing pellets resulting from ethanol precipitation. Tip : Care should be taken when removing the supernatant as pellets from isopropanol precipitation are more loosely attached to the side of the tube.

Tip : Carefully tip the tube with the pellet on the upper side to avoid dislodging the pellet. Tip : For valuable samples, the supernatant can be retained until recovery of the precipitated DNA has been verified. This removes co-precipitated salt and replaces the isopropanol with the more volatile ethanol, making the DNA easier to redissolve.

Tip : Centrifuge the tube in the same orientation as previously to recover the DNA into a compact pellet. Air-dry the pellet for 5—20 min depending on the size of the pellet. Tip : Do not overdry the pellet e. Redissolve the DNA in a suitable buffer. Tip : Use a buffer with a pH of 7. If using water, check pH. Tip : Redissolve by rinsing the walls to recover all the DNA, especially if glass tubes have been used. To avoid shearing the DNA, do not pipet or vortex.

Endotoxin contamination of different plasmid preparation methods The chemical structure and properties of endotoxin molecules and their tendency to form micellar structures lead to copurification of endotoxins with plasmid DNA. How are endotoxins measured? Influence of endotoxins on biological applications Endotoxins strongly influence transfection of DNA into primary cells and sensitive cultured cells, and increased endotoxin levels lead to sharply reduced transfection efficiencies.

Endotoxin-free plasticware and glassware 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. Spectrophotometry DNA concentration can be determined by measuring the absorbance at nm A in a spectrophotometer using a quartz cuvette.

Effects of solvents on spectrophotometric readings Absorption of nucleic acids depends on the solvent used to dissolve the nucleic acid 7. Fluorometry Fluorometry allows specific and sensitive measurement of DNA concentration by use of a fluorescent dye; with common dyes including Hoechst dyes and PicoGreen.

Back to top Restriction endonuclease digestion of DNA Principle of restriction digestion Many applications require conversion of genomic DNA into conveniently sized fragments by restriction endonuclease digestion. Methylation Many organisms have enzymes called methylases that methylate DNA at specific sequences. The CpG dinucleotide occurs about 5 times less frequently in mammalian DNA than would be expected by chance, and most restriction enzymes with a CpG dinucleotide in their recognition site do not cleave if the cytosine is methylated.

Drosophila, Caenorhabditis, and some other species do not possess methylated DNA, and have a higher proportion of CpG dinucleotides than mammalian species. Rare-cutter enzymes therefore cleave more frequently in these species.


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  • Plant DNA is highly methylated, so for successful mapping in plants, choose enzymes that either do not contain a CpG dinucleotide in their recognition site e. The type of digestion affects the ease of downstream cloning: Sticky-ended fragments can be easily ligated to other sticky-ended fragments with compatible single-stranded overhangs, resulting in efficient cloning. Blunt-ended fragments usually ligate much less efficiently, making cloning more difficult. However, any blunt-ended fragment can be ligated to any other, so blunt-cutting enzymes are used when compatible sticky-ended fragments cannot be generated — for example, if the polylinker site of a vector does not contain an enzyme site compatible with the fragment being cloned.

    Compatibility of reaction conditions 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. Setting up a restriction digestion Pipet reaction components into a tube and mix well by pipetting. Tip : Thorough mixing is extremely important. Tip : The enzyme should be kept on ice and added last. Tip : When setting up large numbers of digests, make a reaction master mix consisting of water, buffer, and enzyme, and aliquot this into tubes containing the DNA to be digested.

    Centrifuge the tube briefly to collect the liquid at the bottom. However, some restriction enzymes require higher e. For some downstream applications it is necessary to heat-inactivate the enzyme after digestion. Note that some restriction enzymes are not fully inactivated by heat treatment.

    A typical ligation reaction is set up as per the table Typical ligation reaction. Tip : Simple ligations with two fragments having 4 bp 3' or 5' overhanging ends require much less ligase than more complex ligations or blunt-end ligations. The quality of the DNA will also affect the amount of ligase needed. Higher temperatures make annealing of the ends difficult, while lower temperatures diminish ligase activity.

    Blunt-end ligations require about 10—times more enzyme than sticky-end ligations in order to achieve an equal efficiency. From individual E. Back to top DNA analysis using analytical gels Gels allow separation and identification of nucleic acids based on charge migration. Pouring an agarose gel Agarose concentration The concentration of agarose used for the gel depends primarily on the size of the DNA fragments to be analyzed.

    Add an appropriate amount of agarose depending on the concentration required to an appropriate volume of electrophoresis buffer depending on the type of electrophoresis apparatus being used in a flask or bottle. Tip : The vessel should not be more than half full. Cover the vessel to minimize evaporation. Tip : Always use the same batch of buffer to prepare the agarose as to run the gel since small differences in ionic strength can affect migration of DNA. Heat the slurry in a microwave or boiling water bath, swirling the vessel occasionally, until the agarose is dissolved.

    Tip : Ensure that the lid of the flask is loose to avoid build-up of pressure. Be careful not to let the agarose solution boil over as it becomes super-heated. Tip : If the volume of liquid reduces considerably during heating due to evaporation, make up to the original volume with distilled water. This will ensure that the agarose concentration is correct and that the gel and the electrophoresis buffer have the same buffer composition. Pour the agarose solution onto the gel tray to a thickness of 3—5 mm. Insert the comb either before or immediately after pouring the gel.

    Leave the gel to set 30—40 min. Tip : Ensure that there is enough space between the bottom of the comb and the glass plate 0. Tip : Make sure that there are no air bubbles in the gel or trapped between the wells. Carefully remove the comb and adhesive tape, if used, from the gel. Fill the tank containing the gel with electrophoresis buffer. Tip : Add enough buffer to cover the gel with a depth of approximately 1 mm liquid above the surface of the gel. If too much buffer is used the electric current will flow through the buffer instead of the gel.

    Back to top Running an agarose gel Agarose gel electrophoresis allows analysis of DNA fragments between 0. Gel loading buffer see table Gel loading buffer must be added to the samples before loading and serves three main purposes: To increase the density of the samples to ensure that they sink into the wells on loading. To add color to the samples through use of dyes such as bromophenol blue or xylene cyanol, facilitating loading. To allow tracking of the electrophoresis due to co-migration of the dyes with DNA fragments of a specific size.

    Samples should always be mixed with gel loading buffer prior to loading on a gel. Tip : Do not use sample volumes close to the capacity of the wells, as samples may spill over into adjacent wells during loading. Tip : Be sure that all samples have the same buffer composition. High salt concentrations, for example in some restriction buffers, will retard the migration of the DNA fragments.

    Ensure that no ethanol is present in the samples, as this will cause samples to float out of the wells on loading. Agarose gel electrophoresis Apply samples in gel loading buffer to the wells of the gel. Prior to sample loading, remove air bubbles from the wells by rinsing them with electrophoresis buffer.

    Tip : Make sure that the entire gel is submerged in the electrophoresis buffer. Tip : To load samples, insert the pipet tip deep into the well and expel the liquid slowly. Take care not to break the agarose with the pipet tip. Tip : Be sure to always include at least one lane of appropriate molecular-weight markers. Connect the electrodes so that the DNA will migrate towards the anode positive electrode. Tip : Electrophoresis apparatus should always be covered to protect against electric shocks. This will depend on the size of DNA being analyzed, the concentration of agarose in the gel, and the separation required.

    Tip : Avoid use of very high voltages which can cause trailing and smearing of DNA bands in the gel, particularly with high-molecular-weight DNA. Tip : Monitor the temperature of the buffer periodically during the run. If the buffer becomes overheated, reduce the voltage. Tip : Melting of an agarose gel during the electrophoresis is a sign that the buffer may have been incorrectly prepared or has become exhausted during the run.

    Tip : For very long runs, e.

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    Laboratory Methods in Enzymology: DNA, Volume 529

    Pulse-field gel electrophoresis Apply samples in gel loading buffer to the wells of the gel. Tip : Pulse-field gel electrophoresis uses high voltages, so TBE buffer, which has greater buffering capacity than TAE buffer, should be used. Tip : Prior to sample loading, remove air bubbles from the wells by rinsing them with electrophoresis buffer. Tip : Make sure that the entire gel is submerged in the running buffer. Turn on the power supply and run the gel at V with a switch interval of 5—40 s until the dyes have migrated an appropriate distance. If the buffer becomes heated, reduce the voltage.

    Back to top Visual analysis of the gel Staining To allow visualization of the DNA samples, agarose gels are stained with an appropriate dye. Visualization Ethidium bromide—DNA complexes display increased fluorescence compared to the dye in solution. Denaturation Double-stranded DNA must be denatured in order to create suitable hybridization targets. Neutralization Remove the denaturation buffer and completely cover the gel in neutralization buffer see table Neutralization buffer. Assembling the blotting apparatus Place a support larger than the gel in a tray containing 10x SSC see table 20x SSC , and cover the support with a glass or Plexiglas plate see figure Southern blot setup.

    Cut two lengths of Whatman 3MM paper wider than the gel, long enough to fit under the gel and reach to the bottom of the dish on either side. Wet the sheets briefly in 10x SSC, and place them on the glass plate. Remove any air bubbles between the paper and the support by rolling a pipet several times back and forth over the surface. Cut one sheet of blotting membrane and two sheets of Whatman 3MM paper about 1 mm larger than the gel on each side.

    Tip : Always wear gloves when working with blotting membranes. Handle membranes carefully by the edges or using clean blunt-ended forceps. Place the prepared gel upside-down on the platform. Remove any air bubbles trapped between the gel and the platform by rolling a pipet several times back and forth over the gel. Surround the gel with plastic wrap. This ensures that the 10x SSC moves only through the gel and not around it. Place the precut blotting membrane on top of the gel so that it covers the entire surface. Do not move the blotting membrane once it has been placed on the gel.

    Remove any air bubbles between the paper and the support as described in step 4. Again, remove any trapped air bubbles as described in step 4. Place a 15—20 cm stack of dry paper towels on top of the filter paper. Tip : Make sure that the plastic wrap surrounding the gel prevents contact of the paper towels with the 10x SSC and the wet filter paper under the gel. Ensure that the towels do not droop over since they can cause liquid to flow around the gel instead of through it.

    Place a second glass or Plexiglas plate on top of the paper towels. Place the weight on top of the plate. Let the transfer proceed for 12—18 h. Tip : Transfer efficiency is improved by removing the wet paper towels and replacing them with dry ones at least once during the transfer. Fixing the DNA to the blot After the transfer is complete, remove the weight, paper towels, and the two sheets of filter paper.

    Turn over the gel and the blotting membrane together, and lay them, gel-side up, on a sheet of dry filter paper. Mark the positions of the gel lanes on the membrane using a ballpoint pen or a soft-lead pencil. Peel the gel from the membrane. If desired, keep the gel to assess the efficiency of DNA transfer, otherwise discard.

    Before removing the gel from the blotting membrane, ensure that the gel lanes are marked so that they can be later identified. In order to assess the efficiency of DNA transfer, stain the gel with ethidium bromide after blotting to see how much DNA remains.

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