アガロース ゲル 濃度。 アガロースゲル電気泳動

エムエステクノシステムズ|アガロース

アガロース ゲル 濃度

and are the two most common gel matrices utilized in the electrophoretic separation of nucleic acids. Both materials form 3-dimensional matrices with pore sizes appropriate for separation of nucleic acids and are nonreactive with the samples. The pore sizes can be adjusted by varying the percentage of the matrix, in order to efficiently resolve nucleic acids of different sizes. The choice between agarose and polyacrylamide depends primarily on the size range and desired resolution of separation of nucleic acid samples, although and y methods may be considered Table 1. Agarose forms matrices with pore sizes ideal for separating nucleic acid molecules in the range of 0. 1—25 kb. Polyacrylamide, on the other hand, forms smaller pore sizes, which resolve nucleic acid molecules smaller than 1 kb. , 0. Table 2 provides recommended agarose gel percentages for the separation of DNA fragments of different lengths [2]. In general, higher-percentage gels result in better separation and resolution of smaller fragments Figure 2. Note that low-percentage gels can be fragile and difficult to handle, while high-percentage gels may be turbid and interfere with visualization. Agarose is a purified form of agar, a carbohydrate structural component of the cell wall of marine red algae. The dissaccharide unit, also known as agarobiose, forms a chain connected by a-1,3 linkages Figure 3. When an agarose solution is heated and cooled, it forms a gel matrix with pore sizes ranging from 50 to 200 nm in diameter, as governed by gel concentration. Upon cooling, two agarose chains form helical fibers linked by hydrogen bonds. Further cooling below the gelling point usually 10 kbp, agarose with a may be a better choice. , , where enzymes are active within a semi-solid agarose solution. The powder and liquid forms are known neurotoxins and should be handled with care using protective labwear. The higher the percentage, the smaller the pore size, allowing resolution of smaller molecules. Table 3 shows commonly used gel percentages [2]. 0 100—500 bases 5. 0 70—400 bases 6. 0 40—300 bases 8. 0 30—200 bases 10. 0 20—100 bases 15. 0 10—50 bases 20. 0 5—30 bases 30. 0 1—10 bases Nondenaturing gels 3. 5 100—1,000 bp 5. 0 80—500 bp 8. 0 60—400 bp 12. 0 50—200 bp 15. 0 25—150 bp 20. The crosslinker, bisacrylamide, contains two units of acrylamide joined by a methylene bridge. Agarose and polyacrylamide gels are prepared using an ionic solution with electrical conductivity to enable nucleic acid mobility during electrophoresis. The same buffer type is usually used for both the gel and the running buffer during an electrophoretic run to maintain the same pH and ionic strength. The two most common buffers for nucleic acid electrophoresis are and , both with pH close to neutral to favor negative charges on the nucleic acids learn more:. For analysis of single-stranded DNA or RNA, agarose and polyacrylamide gels are often prepared and run under denaturing conditions. Denaturing conditions disrupt hydrogen bonds that may form between nucleic acids and thus reduce formation of secondary structures such as hairpin loops. Denaturing electrophoresis is therefore more routine for RNA separation and analysis. Common denaturing buffers used with agarose and polyacrylamide gels in nucleic acid electrophoresis include:• Agarose: Glyoxal and DMSO in sodium phosphate buffer, NaOH-EDTA buffer, and formaldehyde or in MOPS buffer• Polyacrylamide: in TBE buffer• Ladder type i. , DNA or RNA , fragment structure i. , single-stranded or double-stranded , and conformation i. , supercoiled, open circular, or linear , to ensure appropriate comparisons of migration learn more:• Number of fragments and their separation patterns for proper size estimation• Intended uses, such as whether the ladder is designed for qualitative analyses vs. accurate• Suitability of the ladder for the type of gel used e. , some precast gels recommend designed for optimal runs• Nature of loading dyes, to g the bands of interest Figure 6• Compatibility of loading buffer with the gel used e. , salt concentration of the buffer may impact migration of the sample In the early days, DNA size standards were primarily derived from restriction digest fragments of viral genomes e. , and bacterial plasmids e. These standards often had issues with reproducibility of digestion, sample purity, and banding patterns in electrophoresis. Today, chromatographically purified fragments are considered the gold standard for ladders, since the technology provides higher control over quality, banding pattern, intensity, and quantity Figure 6. Today, ladders are also designed for , for both shipment and storage, and for convenience and reduced environmental impact. Premixed, ready-to-use ladders are also , where the ladders have been prepared with an optimal concentration of the loading dye to load directly onto the gels. Note that ladders from different manufacturers with the same description e. , 1 kb or 100 bp may not contain the same number, size, and intensity of DNA fragments Figure 7. In contrast to DNA ladders, are usually provided with a loading buffer containing a denaturant. Denaturants help maintain RNA in single-stranded form, allowing more and separation results. When performing RNA gel electrophoresis, DNA ladders should be avoided, because their use under denaturing conditions can lead to atypical separation patterns due to separation of the double strands. Figure 6. Differences in DNA ladders. Ladder 2 consists of chromatographically purified DNA fragments in an optimal loading buffer, resulting in bands of equal or desired intensity, lack of band smears, and absence of dye shadows. In contrast, ladder 1 was manufactured with an older technology and loaded in a buffer with suboptimal composition. The amount of DNA that should be loaded onto the gel must be calculated to ensure the bands of interest are well separated for visualization and detection. Note that overloading a sample or standard can result in smearing of bands and masking those nearby, resulting in poor resolution, particularly when the fragments are of similar sizes Figure 8A. Using a smaller volume may result in band distortion, due to poor distribution in the well Figure 8B. For samples containing DNA-binding proteins or cohesive ends, the mixture may need to be heated in a prior to gel loading, because protein binding and interaction between the DNA fragments may cause poor separation Figure 10B. typically made as 6X or 10X stock solutions are added to the samples and the standard, when needed in preparation for gel electrophoresis. Components of loading buffers include the following:• A density ingredient, such as glycerol or sucrose, increases viscosity of the samples, ensuring that the samples sink into the wells. Salts, such as Tris-HCl, create environments with favorable ionic strength and pH for the samples. Loading buffers with high salt concentrations may produce broader or distorted bands and smears. A metal chelator, such as EDTA, prevents nucleases present in the sample from degrading nucleic acids. Dyes provides color for easy monitoring of sample loading, progress of the electrophoretic run, and often pH changes. Some loading buffers may contain more than one dye, to track migration of molecules of varying sizes in a sample more efficiently. Typically, loading dyes are small and negatively charged molecules so that they migrate in the same direction as the nucleic acids. Some display pH-dependent colors, serving as pH indicators for samples during loading and running Figure 9A. Commonly used dyes include bromophenol blue, xylene cyanol, phenol red, and orange G. When choosing a loading buffer, pay attention to the apparent migration of the dye s Figure 9B, Tables 5 and 6 to avoid masking the nucleic acid bands of interest, especially if they have similar molecular sizes Figure 9C. Dye masking makes analysis and quantitation of the desired bands problematic and less reliable. At times the loading buffer includes detergents or reducing agents, such as , , and , for denaturing. These additives can disrupt molecular interactions between and within nucleic acid molecules, promoting linearity or single-stranded conformation of the molecules. Samples should be heated with denaturants in loading dye to obtain optimal separation results Figure 10A. For the electrophoresis of double-stranded DNA from enzymatic reactions, SDS may be added to the loading buffer to disrupt interactions between proteins and nucleic acids, in order to prevent alteration of sample mobility Figure 10B. Figure 10. Effects of heat and SDS on electrophoresis of samples. A RNA ladders in a denaturing buffer were loaded onto the gel without heat treatment. B DNA samples from a restriction digest and a ligation reaction were prepared in loading buffer with or without SDS. Samples in SDS were heated before gel loading. 5 750 bp 1,150 bp 13,000 bp 16,700 bp 0. 6 540 bp 850 bp 8,820 bp 11,600 bp 0. 7 410 bp 660 bp 6,400 bp 8,500 bp 0. 8 320 bp 530 bp 4,830 bp 6,500 bp 0. 9 260 bp 440 bp 3,770 bp 5,140 bp 1. 0 220 bp 370 bp 3,030 bp 4,160 bp 1. 2 160 bp 275 bp 2,070 bp 2,890 bp 1. 5 110 bp 190 bp 1,300 bp 1,840 bp 2. 0 65 bp 120 bp 710 bp 1,040 bp 3. 0 30 bp 60 bp 300 bp 460 bp 4. 0 18 bp 40 bp 170 bp 260 bp 5. 0 50 bases 230 bases 5. 0 35 bases 130 bases 6. 0 26 bases 105 bases 8. 0 19 bases 75 bases 10. 0 12 bases 55 bases 15. 0 10 bases 40 bases 20. 0 8 bases 28 bases 30. 0 6 bases 20 bases Nondenaturing gels 3. 5 100 bp 460 bp 5. 0 65 bp 260 bp 8. 0 45 bp 160 bp 12. 0 20 bp 70 bp 15. 0 15 bp 60 bp 20. 0 12 bp 45 bp Electrophoresis is carried out following the preparation of gels, standards, and samples. The gel must be completely solidified before removal of the comb and addition of the running buffer. The should be lifted upward, smoothly and steadily, to avoid tearing the gel and distorting the wells. After buffer addition and comb removal, care should be taken to remove air bubbles that may be trapped in the wells. For polyacrylamide gels, the wells should be thoroughly rinsed with buffer to remove residual unpolymerized acrylamide. Horizontal gels should be oriented in a so that sample wells are on the side of the negative electrode to move samples towards the positive electrode when electrophoresis starts Figure 11A. Vertical gel boxes are designed with wells located at the top Figure 11B. A running buffer, which is an ionic solution with buffering capacity, is routinely used in gel runs to allow current flow while impeding pH changes that may occur. During electrophoresis, the negative electrode becomes more basic and the positive electrode more acidic because of electron flow, resulting in electrolysis of water and shifts in pH Table 6. Release of hydrogen and oxygen gases causes bubbling from the electrodes, a telltale sign of a running gel. Ideally, the running buffer and the buffer should be the same to ensure efficient conductivity. The choice of buffer for electrophoresis depends on sample sizes, run time, and post-electrophoresis processes, with Tris-acetate EDTA TAE and Tris-borate EDTA TBE being the two most common buffers Table 7 [2,7]. Due to its lower buffering capacity, TAE is more suitable for shorter electrophoretic runs e. has a higher buffering capacity, making it less likely to overheat and thus better suited for longer runs. TBE works better for the separation of shorter fragments, but dsDNA may migrate more slowly in TBE. TBE can inhibit enzymes and therefore may not be suitable for downstream applications involving enzymatic steps, such as , , and. Using a buffer with ionic strength higher than 1X TAE or 0. 5—1X TBE may move the samples faster but likely will generate a large amount of heat due to high conductivity, resulting in sample denaturation and damage to the gel. Buffer Advantages Disadvantages Nucleic acid resolution DNA RNA• Better separation of large fragments• Low buffering capacity; better for shorter runs• Good for downstream enzymatic applications• High ionic strength and buffering capacity; good for longer runs• Less prone to overheating• Inhibits enzymes, making it unsuitable for downstream enzymatic steps e. To start running a gel, an electrical potential is applied across the gel with constant voltage, current, or power learn more:. Recommended voltages are usually provided with commercially available , for optimal separation of the fragments in each product. Note that very low voltage slows the migration of the nucleic acids, which may result in diffusion of small molecules and low resolution Figure 13A. In some cases, a temperature probe may be connected to the gel apparatus to help control cooling and heating of the buffer. The length of the gel, the voltage used, and the sizes of the molecules in the sample will determine the amount of time needed for electrophoresis. Most importantly, run time should be monitored to ensure the smallest molecules in the samples or standards do not migrate off the gel. Note that run times shorter than necessary will not be sufficient to completely resolve the bands Figure 14. DNA ladders containing are available to help monitor gel runs, as well as to ensure that bands of interest are not masked by the dyes. After a gel run is complete, the samples need to be visualized. Since nucleic acids are not visible under ordinary ambient lighting, a detection method is required for visualization. As described in Table 9, available methods offer differing ranges of sensitivity and benefits in sample detection [6]. Stain Benefits and considerations Sensitivity for detection approximate dsDNA amounts Colorimetric• Methylene blue• Crystal violet• Requires no special equipment• Requires a destaining step• Low sensitivity 0. Often mutagenic• May require special waste disposal• Offers shorter workflows with high sensitivity• May be used to stain samples electrophoresis 25 pg—1 ng Radioactive• 32P• 33P• Requires training and precautions for radioactive safety• Detects or labels samples by radioactive probes or nucleotides• Common for detection of oligonucleotides• Considered most sensitive 10 fg—1 ng Among available stains, fluorescent dyes are most widely utilized in sample detection Figure 15 due to their ease of use and high sensitivity. When excited with an appropriate wavelength, the dyes emit visible light i. , fluoresce learn more:. The intensity of fluorescence correlates to the amount of nucleic acid bound, which is the basis for detection and quantitation of nucleic acids in electrophoresis. Mutagenicity and disposal: Ethidium bromide is highly mutagenic, so fluorescent dyes that are offer a safer workflow in nucleic acid electrophoresis Figure 16. Sensitivity: Fluorescent dyes that are than EtBr are better suited for detection of low amounts of samples Figure 16. Therefore, dyes that are good alternatives for sample detection in RNA electrophoresis. UV damage: Fluorescent dyes that can be excited with instead of UV light cause less structural damage to nucleic acids but detect with equal or higher sensitivity Figure 17A. As such, excitation with blue light in electrophoresis can improve success in downstream applications like and Figure 17B. Figure 17. A Excitation and emission spectra of common nucleic acid stains. The stains are excited most efficiently at a particular wavelength called the excitation maximum. and stains can be excited maximally by and to a lower extent by UV light. B Cloning efficiency after indicated exposure times using blue light for SYBR Safe stain or UV light for ethidium bromide , for visualization of the cloning insert during electrophoresis. Cloning efficiency of the gel-purified lacZ fragment was measured by the number of blue colonies formed on the plates, which indicates insertion of the functional unmutated gene into the vector. In place of staining with a dye, nucleic acids may be indirectly visualized by a method call UV shadowing, taking advantage of UV absorption by nucleic acids [8]. For detection by UV shadowing, nanograms to micrograms of samples are needed, and a thin and transparent gel like polyacrylamide should be used to ensure UV absorption and transmission. In a UV shadowing protocol, the gel is removed from the cassette after electrophoresis to maximize detection, wrapped in clear plastic film for protection, and then placed on a UV-fluorescent thin layer chromatography TLC plate. When the gel is exposed to UV radiation, absorption by the nucleic acid bands casts shadows on the TLC plate Figure 18. The shadowy areas of the gel of desired sizes are cut out for further processing. After visualization, nucleic acid gels are typically documented for record and analysis of electrophoresis results. If samples are stained with a fluorescent dye, special equipment is required to the dye with an appropriate light source to both visualize and capture a gel image. An excitation light source may be above the gel, called an epi-illuminator similar to a handheld UV lamp , or below the gel, called a transilluminator Figure 19. Since the light source is farther in the epi-illuminator setting, the samples are subjected to less energy. This can lessen UV damage to the nucleic acids but may also lower the signal of the gel bands. A transilluminator, on the other hand, provides higher signal for the bands but may increase UV-induced damage due to the proximity of the radiation to the gel. In conclusion, nucleic acid electrophoresis workflows employ a number of steps and reagents to separate and analyze samples. Choosing the right tools for your samples, as well as recognizing workflow advantages and disadvantages, can significantly improve the results of electrophoresis in molecular biology applications. Popular• Popular• Most Popular Products• Most Popular Categories• Order Tools• Product Documentation• Other Product Information• Product Support• Educational Resources•

次の

アガロースゲル電気泳動

アガロース ゲル 濃度

電気浸透度が低いため、短い電気泳動距離でも良好な分離能が得られます。 1 Hinc II digest lane 2: 0. 5ug, lane 3: 0. 2ug, lane 4: 0. 1ug, lane 5: 0. 分解能は、アクリルアミドに匹敵し、200-1,000bpの範囲における分離可能なフラグメント差は、フラグメント全体長の約2%です。 ゲル強度に優れているため、取り扱いに大変便利です。 0~7. in-gel反応とバンドの切り出しにはTAEバッファーがより適しています。 バックグラウンドが低く、スメアにならず正確なバンドパターンを示します。 ゲル強度が高く取り扱いし易いので、ブロッティング用途に最適です。

次の

電気泳動関連:Q&A |タカラバイオ株式会社

アガロース ゲル 濃度

アガロースゲル電気泳動(アガロースゲルでんきえいどう、 : agarose gel electrophoresis)は、(の主成分)を使用したにより、をその大きさに応じて分離する手法。 数ある電気泳動の中でも、もっともオーソドックスなものといえる。 原理 、などの核酸分子はそれぞれ固有の大きさ(長さ)とを持っている。 核酸の場合は荷電の個数はの大きさに比例するため、長さ当たりの駆動力はほぼ一定である。 しかし、ゲルを構成するアガロース分子の網目と絡みながら移動しなければならないため、長い核酸分子ほど移動速度が遅くなる。 またゲルのアガロース濃度が高いほど移動速度が遅くなるので、分離したい核酸のサイズに合わせてゲルのアガロース濃度が選択される。 手法 適切なに高純度のアガロースを加え、加熱して溶かした後、型枠に入れて固める。 アガロースの量は、目的に応じて0. このときあらかじめ、櫛(くし)状のプラスチック片(コームと呼ばれる)を利用して核酸溶液を注入するための穴(wellと呼ばれる)を一方の端に作る。 固まったゲル片を緩衝液の入った水槽に入れ、核酸溶液を注入した後、一方からをかける。 一定時間後、ゲル片を取り出し、などの核酸染色溶液に浸漬する。 染色薬剤をあらかじめ元の核酸溶液やアガロースゲルに添加しておく場合もある。 染色された核酸はを照射するとを発するので、その存在が確認できる。 また染色に使われた臭化エチジウムの量はDNAの分子の長さと量に比例するため、蛍光の強さによってDNA量を確認することができる。 主な用途 から核酸(DNAやRNA)を抽出した際、その確認のためにアガロースゲル電気泳動を行う。 また、によりDNAを増幅したときにも、確認に使用される。 その後、や、ゲル抽出などを行うこともある。 プラスミドベクターに組み込んだ目的の遺伝子を確認するため、処理を行った後、電気泳動で確認することもある。 この項目は、に関連した です。 などしてくださる(/)。

次の