In molecular biology, gel extraction or gel isolation is a technique used to isolate a desired fragment of intact DNA from an agarose gel following agarose gel electrophoresis. After extraction, fragments of interest can be mixed, precipitated, and enzymatically ligated together in several simple steps. This process, usually performed on plasmids, is the basis for rudimentary genetic engineering. After DNA samples are run on an agarose gel, extraction involves four basic steps: identifying the fragments of interest, isolating the corresponding bands, isolating the DNA from those bands, and removing the accompanying salts and stain. To begin, UV light is shone on the gel in order to illuminate all the ethidium bromide-stained DNA. Care must be taken to avoid exposing the DNA to mutagenic radiation for longer than absolutely necessary. The desired band is identified and physically removed with a cover slip or razor blade. The removed slice of gel should contain the desired DNA inside. An alternative method, utilizing SYBR Safe DNA gel stain and blue-light illumination, avoids the DNA damage associated with ethidium bromide and UV light.[1] Several strategies for isolating and cleaning the DNA fragment of interest exist. Spin Column Extraction[edit]Gel extraction kits are available from several major biotech manufacturers for a final cost of approximately 1–2 US$ per sample. Protocols included in these kits generally call for the dissolution of the gel-slice in 3 volumes of chaotropic agent at 50 °C, followed by application of the solution to a spin-column (the DNA remains in the column), a 70% ethanol wash (the DNA remains in the column, salt and impurities are washed out), and elution of the DNA in a small volume (30 µL) of water or buffer.[2] Dialysis[edit]The gel fragment is placed in a dialysis tube that is permeable to fluids but impermeable to molecules at the size of DNA, thus preventing the DNA from passing through the membrane when soaked in TE buffer. An electric field is established around the tubing (in a way similar to gel electrophoresis) long enough so that the DNA is removed from the gel but remains in the tube. The tube solution can then be pipetted out and will contain the desired DNA with minimal background. Traditional[edit]The traditional method of gel extraction involves creating a folded pocket of Parafilm wax paper and placing the agarose fragment inside. The agarose is physically compressed with a finger into a corner of the pocket, partially liquifying the gel and its contents. The liquid droplets can then be directed out of the pocket onto an exterior piece of Parafilm, where they are pipetted into a small tube. A butanol extraction removes the ethidium bromide stain, followed by a phenol/chloroform extraction of the cleaned DNA fragment. The disadvantage of gel isolation is that background can only be removed if it can be physically identified using the UV light. If two bands are very close together, it can be hard to separate them without some contamination. In order to clearly identify the band of interest, further restriction digests may be necessary. Restriction sites unique to unwanted bands of similar size can aid in breaking up these potential contaminants. References[edit]
For gel extraction/cleanup of up to 10 μg DNA (70 bp to 10 kb) from enzymatic reactions ✓ 24/7 automatic processing of online orders ✓ Knowledgeable and professional Product & Technical Support ✓ Fast and reliable (re)-ordering QIAquick Gel Extraction Kit (1000) Cat. No. / ID: 28706X4 For gel extraction or cleanup of 1000 reactions: 1000 QIAquick Spin Columns, Buffers, Collection Tubes (2 ml) $2,099.00Log in to see your account pricing. KitColumn QIAquick Gel Extraction Kit QIAquick PCR & Gel Cleanup Kit The QIAquick Gel Extraction Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease. ✓ 24/7 automatic processing of online orders ✓ Knowledgeable and professional Product & Technical Support ✓ Fast and reliable (re)-ordering Features
Product DetailsThe QIAquick Gel Extraction Kit provides spin columns, buffers, and collection tubes for silica-membrane-based purification of DNA fragments from gels (up to 400 mg slices) or enzymatic reactions. DNA ranging from 70 bp to 10 kb is purified using a simple and fast bind-wash-elute procedure and an elution volume of 30–50 μl. An integrated pH indicator allows easy determination of the optimal pH for DNA binding to the spin column. The procedure can be fully automated on theQIAcube Connect. For optimal results it is recommended to use this product together with QIAvac 24 Plus. PerformanceThe QIAquick Gel Extraction Kit enables removal of nucleotides, enzymes, salts, agarose, ethidium bromide, and other impurities from samples, ensuring up to 80% recovery of DNA (see figure " High recoveries from gels"). Using a microcentrifuge or vacuum manifold, DNA ranging from 70 bp to 10 kb is purified from 1–24 samples. Purified DNA can be used, for example, in sequencing (see figure " Reliable sequencing after gel extraction"). DNA fragments smaller than 70 bp or larger than 10 kb should be extracted with the QIAEX II Gel Extraction System. See figuresPrincipleQIAquick Kits contain a silica membrane assembly for binding of DNA in high-salt buffer and elution with low-salt buffer or water. The purification procedure removes primers, nucleotides, enzymes, mineral oil, salts, agarose, ethidium bromide, and other impurities from DNA samples (see figure " High recoveries from gels"). Silica-membrane technology eliminates the problems and inconvenience associated with loose resins and slurries. Specialized binding buffers are optimized for specific applications and promote selective adsorption of DNA molecules within particular size ranges. Gel loading dyeTo enable faster and more convenient sample processing and analysis, gel loading dye is provided. GelPilot Loading Dye contains three tracking dyes (xylene cyanol, bromophenol blue, and orange G) to facilitate the optimization of agarose gel run time and prevent smaller DNA fragments migrating too far (see figure "GelPilot Loading Dye"). See figuresProcedureThe QIAquick system uses a simple bind-wash-elute procedure (see flowchart " QIAquick and MinElute procedure"). Gel slices are dissolved in a buffer containing a pH indicator, allowing easy determination of the optimal pH for DNA binding, and the mixture is applied to the QIAquick spin column (see figure " pH Indicator Dye"). Nucleic acids adsorb to the silica membrane in the high-salt conditions provided by the buffer. Impurities are washed away and pure DNA is eluted with a small volume of low-salt buffer provided or water, ready to use in all subsequent applications. HandlingQIAquick spin columns are designed to provide two convenient handling options. The spin columns fit into a conventional table-top microcentrifuge or onto any vacuum manifold with luer connectors, such as the QIAvac 24 Plus with QIAvac Luer Adapters. The QIAquick Gel Extraction Kit, in addition to other QIAGEN spin-column-based kits, can be fully automated on the QIAcube, enabling increased productivity and standardization of results (see figures "Spin column handling options A, B, C, D, and E"). See figuresApplicationsDNA fragments purified with the QIAquick system are ready for direct use in all applications, including sequencing, ligation and transformation, restriction digestion, labeling, microinjection, PCR, and in vitro transcription. Supporting data and figuresQIAquick and MinElute procedure.The QIAquick and MinElute systems use a simple bind-wash-elute procedure with spin columns or a vacuum manifold. Specifications
ResourcesPublicationsFAQLarger DNA fragments bind more tightly to the QIAquick columns. It is difficult to predict whether a DNA fragment larger than 10 kb can be efficiently recovered, because this depends on base composition as well as fragment size. If the fragment is only a few kb larger than the 10 kb limit, it can be helpful to heat the elution buffer EB to 60°C and let it incubate on the column for a few minutes before centrifuging. However, please note that it will become less likely to recover your sample the larger the fragment size is. As we cannot guarantee recovery of fragments larger than the maximum cutoff size, we do not recommend to purify such fragments using QIAquick Kits. The QIAEX II Kit can be used to extract DNA fragments up to 50 kb from agarose or polyacrylamide gels. FAQ ID -756 Buffer PB of the QIAquick PCR Purification Kit cannot be used to extract DNA from agarose gels. However, Buffer QG of the QIAquick Gel Extraction Kit can be used to remove salt and proteins from enzymatic reactions by adding 3 volumes of Buffer QG and 1 volume of isopropanol to the reaction and proceeding with step 6 of the Gel Extraction Spin Protocol in the QIAquick Spin Handbook. See the QIAquick Spin Handbook for a list of reactions which can be cleaned up with the various QIAquick kits. FAQ ID -786 No. While columns from the QIAprep Spin Miniprep Kit and the QIAquick PCR Purification- and Gel Extraction Kits are based on silica-gel-membrane technology, each is designed to work optimally within its own kit format. In addition, the binding capacity and DNA recovery size cut-offs of the QIAprep and QIAquick Spin columns are different. QIAprep Spin columns bind up to 20 ug of plasmid DNA up to 50 kb in length, while the QIAquick Spin columns bind up to 10 ug of DNA with a maximum fragment size of 10 kb. FAQ ID -311 Yes - all QIAquick Spin Kits contain identical columns, but different binding buffers optimized for each specific application. FAQ ID -577 Cut out the slice of agarose containing the DNA fragment of interest, and store it at 4oC in an Eppendorf tube sealed with Parafilm. FAQ ID -313 Buffer PE did not contain ethanol Ethanol must be added to Buffer PE (concentrate) before use. Repeat procedure with correctly prepared Buffer PE. Inappropriate elution buffer DNA will only be eluted efficiently in the presence of low-salt buffer (e.g., Buffer EB: 10 mM Tris·Cl, pH 8.5) or water. Elution efficiency is strongly dependent on the salt concentration and pH of the elution buffer. Contrary to adsorption, elution is most efficient under basic conditions and low salt concentrations. DNA is eluted with 50 or 30 µl of the provided Buffer EB (10 mM Tris·Cl, pH 8.5), or water. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water to elute, make sure that the pH is within this range. In addition, DNA must be stored at –20°C when eluted with water since DNA may degrade in the absence of a buffering agent. Elution with TE (10 mM Tris·Cl, 1 mM EDTA, pH 8.0) is possible, but not recommended because EDTA may inhibit subsequent enzymatic reactions. Elution buffer incorrectly dispensed Add elution buffer to the center of the QIAquick membrane to ensure that the buffer completely covers the membrane. This is particularly important when using small elution volumes (30 µl). FAQ ID -180
FAQ ID -305 Based on our experience, the QIAquick PCR Purification Kit removes SYBR Green dye efficiently from PCR reactions. We strongly assume that the QIAquick Gel Extraction Kit will be equally efficient in removing this dye, however, we recommend a 5 min incubation with wash Buffer PE on the QIAquick spin column at step 10 of the QIAquick Gel Extraction Kit Protocol. Efficiency of SYBR Green dye removal has to be validated by the enduser. FAQ ID -637 Yes, please follow the Supplementary Protocols 'High-throughput gel extractions using the QIAquick 96 PCR Purification Kit' (QQ03). Please contact your local QIAGEN Technical Service for this protocol. FAQ ID -944 Yes. The QIAquick Gel Extraction Kit for extraction of DNA from gels can also be used for RNA gel extraction. Please see a user-developed procedure below, which was kindly provided by J. Knobloch, Heinrich Heine University, Düsseldorf, Germany. Note that this protocol has not been thoroughly tested and optimized by QIAGEN. QIAquick Gel Extraction Kits are not guaranteed to be RNase-free.
Figure 1: A. Total RNA was isolated from the parasitic blood fluke Schistosoma mansoni using the RNeasy Mini Kit and run on a formaldehyde agarose (1.2%) gel. (Note: The 28S rRNA in S. mansoni contains a break site so that the rRNA splits into two parts, which run on a gel at the same size as the 18S rRNA.) The rRNA bands were excised and treated as described above (left lane) or using 10 volumes of Buffer QG in step 3 (right lane). B. The extracted RNA was then analyzed on a new formaldehyde agarose gel. (Data kindly provided by J. Knobloch, Department of Genetic Parasitology, Heinrich Heine University, Düsseldorf, Germany). FAQ ID -133 DNA fragments purified with the QIAGEN DNA Cleanup Systems, i.e., the QIAquick PCR Purification Kit, the MinElute Reaction Cleanup Kit, the QIAEX II Gel Extraction Kit etc. may float out of the loading wells of agarose gels due to residual ethanol carried over from the wash step with Buffer PE (despite the addtition of glycerol-containing loading buffer). Use either of the following options to remove residual ethanol from the eluate:
FAQ ID -205 Yes, bisulfite containing methylation reactions can be cleaned up with our silica-based cleanup products, such as QIAquick and QIAEX II. Please see Goyon et al. (1994), 'Perpetuation of cytosine methylation in Ascobolus immersus implies a novel type of maintenance methylase', published in J Mol Biol. 1994 Jul 1;240(1):42-51, for a reference. FAQ ID -519 As a rule of thumb, single-stranded DNA binds to silica with approximately half the affinity of a double-stranded DNA fragment of the same length under the buffer conditions used in the QIAquick and MinElute Kits. Even though no systematic experimental data exists, we expect that recovery of ssDNA fragments of approximately 200 nucleotides and below will not be very efficient after cleanup using the QIAquick PCR Purification Kit or MinElute PCR Purification Kit. By comparison, it should be possible to purify fragments longer than 140 nucleotides using the QIAquick Gel Extraction Kit. Note that recovery of single strand DNA is influenced to some degree also by factors such as base composition and secondary structure. It has to be determined empirically by the researcher if cleanup of single-stranded DNA with QIAquick columns yields satisfactory results. FAQ ID -759 Occasionally, DNA fragments eluted from the silica matrix of QIAquick, MinElute or QIAEX II Kits will contain denatured single-stranded DNA (ssDNA), appearing as a smaller band on an analytical gel. Under certain conditions, chaotropic agents (present in all silica-based DNA purification methods) can denature DNA fragments. This is a rare event that may be influenced by sequence characteristics such as the presence of inverted repeats or A–T-rich stretches. Because salt and buffering agents promote renaturation of DNA strands, the following tips are recommended:
FAQ ID -148 Always dispose of potentially
biohazardous solutions according to your institution’s waste-disposal guidelines. Although the lysis and binding buffers in QIAamp, DNeasy, and RNeasy kits contain chaotropic agents that can inactivate some biohazardous material, local regulations dictate the proper way to dispose of biohazards. DO NOT add bleach or acidic solutions directly to the sample-preparation waste. Guanidine hydrochloride in the sample-preparation waste can form highly reactive compounds when combined with
bleach. FAQ ID -12 The QIAEX II and QIAquick Gel Extraction Kit can be used to extract DNA from polyacrylamide gels. The QIAEX II Handbook contains a protocol for Polyacrylamide Gel Extraction. A specialized User-Developed Protocol (QQ05) is available when using the QIAquick Gel Extraction Kit for this purpose. Both protocols require the preparation of a diffusion buffer and a disposable plastic column or syringe barrel containing a Whatman GF/C filter or siliconized glass wool. To ensure optimal diffusion, cut the gel slices as small as possible, and use 2 volumes of diffusion buffer per 1 volume of gel. Increasing incubation time (protocol step 3) may result in higher yields. FAQ ID -120 |
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