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DNA-Isolation Kits Products from Genomed, Germany
TAG Copenhagen A/S offers the full range of products from a leading company in the field of DNA isolation kits:
Jetstar 2.0 Jet star 2.0 is a new unique anion exchange resin developed by Genomed to purify plasmid DNA from E.coli cells. It opens up new dimensions for the preparation of nucleic acids. This new technology i completely non-toxic. The use of phenol, chloroform, CsCl and ethidium bromide is avoided in all Genomed protocols. The JETSTAR 2.0 resin is supplied in disposable mini, midi an maxi columns. These fast-flow columns are used under gravity flow conditions without further instrumentation. All components of the jetstar 2.0 kits are ready to use.
JetQuick Plasmid Miniprep Spin Kit Genomed has developed the jetquick product line on the basis of ultra quick micro spin columns. The new jetquick Plasmid Miniprep Spin kits provide a very simple and reliable method to achieve highly pure plasmid DNA from E.coli cultures.
JetSpin JetSpin The jetspin plasmid kits provide a vary simple and reliable method to isolate highly pure high copy plasmid DNA on the basis of spin columns. The entire procedure is completed in 25 min or less, with ready-to-use DNA in water or TE buffer. No organic extraction or alcohol precipitation is required. The Jetspin Columns are the core of these plasmid kits. They contain a fibrous,polymer-coated matrix. This mew and unique approach leads to very high capacities and an excellent purity of the prepared plasmid DNA.
Jetprep introduction The extraction and purification of Plasmid DNA form E.coli cells is one of the most tedious and time consuming, yet necessary procedures in molecular biology. This is especially true when large numbers of small-scale plasmid preparations (minipreps) are required. Therefore, Genomed has developed the jetprep plsamid Miniprep kit, which provides a simple, reliable and cost-saving method to isolate plsamid DNA.
Jetsorb intorduction The jetsorb gel extraction kit is designed to extract 20 bp - 45.000 bp DNA fragments form all types of agarose gels, including TBE gels. Moreover, the DNA extraction can be carried out form LMP (low melting point) abarose as well as HMP (high melting Point) agarose. Jetsorb is the core-element of the gel extraction kit. It consists of a high quality chromatography material based om spherical 4µm silica gel particles. The surface of the beads has been modified to obtain the unique characteristics of jetsorb in DNA extraction and purification. The Jetsorb gel extraction kit fulfills all the demands of a high quality product in this field. It is the ideal tool to purify all types of DNA fragments, e.g. PCR fragments and restriction fragments. Jetsorb procedure
Jetquick Gel Extraction Spin Kit introduction Genomed has developed the jetquick product line on the basis of ultra quick micro spin columns. The new jetquick gel extraction kit provides a very simple and reliable method to achieve highly pure DNA fragments from high melting point and low melting point agarose. TAE, TBE and other electrophoresis buffer systems can be used.
Jetquick PCR Purification Spin Kit introduction The polymerase chain reaction (PCR) has rapidly established itself as one of the most widely used techniques in molecular biology. It is commonly used in processes as sequencing, cloning, probe preparation, clone screening and mapping. But in many cases a post-purification of the DNA fragment to get rid of primers, nucleotides, polymerases and salts, is required. Genomed has developed the jetquick product line on the basis of ultra quick micro spin columns. The new Jetquick PCR Purification Spin Kit provides a very simple and reliable method to achieve ultrapure DNA fragments (PCR products) from PCR assays.
Jetpure Introduction The Jetpure PCR product purification kit is the ideal tool to purify and concentrate double-stranded PCR products directly form the PCR assay. Jetpure is a high quality chromatography support which is based om a new resin. It is especially developed for the easy separation of double-stranded DNA (PCR product) from single-stranded oligoucleotides (primers) and proteins. Jetpure Procedure The procedure starts directly from the PCR assay. It is a simple and quick procedure which follows the very easy "bind-wash-elute" principle. One of the main jetpure characteristics is the ability to separate double-stranded DNA fragments form single- standed oligonucleotides. This can bi demonstrated by the effective removal of very large amounts of oligonucleotides form a mixture of DNA fragments. The DNA fragments were very different in length with just a few ng per single band. More than 99.5% of the single-stranded oligonucleotides were removed. Jetnick introduction Radiolabling of DA using 32P and 35S-nucleotides is still a very significant technique, when labelled DNA fragments for sequencing, mapping and screening of recombinant clones are required. For this purpose, the jetnick probe purification kit to purify DNA fragments after in vitro labelling reactions was designed. The kit contains a surface modified higt-tec resin with an extremely high affinity to DNA, but hardly any to salts, proteins og other macromolecules. The chromatography resin is combined with a finely tuned procedure which fulfills all demands of quality, convenience and quickness.
Jetquick Introduction Genomed has developed the jetquick DNA clean-up spin kit on the basis of ultra quick micro spin columns. The product is designed to extract and purify linearized DNA fragments, plasmid DNA etc. from solutions, such as restriction assays, ligation assays etc. This post-purification of DNA removes nucleotides, enzymes, salts, dyes and other impurities. The new jetquick DNA clean up spin kit provides a very simple and reliable method to achieve highly pure DNA in minutes. Jetquick procedure The principle of the procedure is to adjust the assay (restriction assay, ligation assay etc.) to defined salt condition an to apply the so treated assay directly onto the jetquick micro spin column. The DNA is selectively bound to the highly specified silica membranes during centrifugation. The jetquick membrane can adsorb up to 20 µg of double stranded DNA per spin column. After centrifugation, one wash is sufficient to remove residual primers, nucleotides, enzymes, salts and other impurities. The jetquick purified DNA is eluted in TE buffer and water. The Jetquick procedure is completed in 5 min. The purified DNA is now ready to use for a large number of applications. JETWELL 96-well PCR Product Purification system Introduction The JETWELL 96-well PCR product purification system is for the simultaneous purification of 96 PCR assays. Each well has a DNA capacity of ³20 µg. The DNA is highly pure and suitable for all enzymatic in vitro applications, like fluorescent sequencing, radioactive sequencing, restriction enzyme digestion, cloning procedures and microarray analysis. The kit doesn’t use any toxic reagents like phenol, chloroform or ethidium bromide and yields DNA that is directly eluted in Tris or another low-salt buffer, thus being ready-to-use. All components should be at room temperature when used. The Principle The JETWELL 96-well PCR purification system combines the convenience of multiwell technology with the selective binding properties of a proprietary silica membrane. The binding conditions provided with the kit are optimized for the efficient recovery of DNAs ranging in size from 80 bp to 20 kb. Oligo primers with a length of up to 40 nucleotides, unincorporated dNTP’s, polymerases and salts are removed to an extent of ³99.5%. The JETWELL 96-well plate procedure The PCR product mixed with binding buffer is loaded into the particular wells of the 96-well plate and processed over the silica membrane at the bottom of each well by centrifugation. The system is compatible with gelatin, detergents or DMSO present in the PCR reaction buffer, as well as with oil or paraffin overlays. There is no need to remove oil overlays before mixing with the binding buffer H1. During the processing over the membrane the DNA will bind reversibly to the silica. After the removal of the unwanted components (i.e. oligos, unincorporated dNTP’s, salts, DNA polymerases) during the following washing step(s), the DNA is eluted in a low-salt buffer (10 mM Tris-Cl [pH 9.0], provided). The isolated DNA can directly be used for downstream applications. Protocol The current protocol is for a centrifuge-driven 96-well procedure: Preliminary steps 1.) Reconstitute buffer H2 with absolute ethanol as described on the bottle’s label. 2.) Be careful when handling buffer H1 as this buffer contains guanidine hydrochloride. This substance is an irritant. Wear gloves and goggles when handling this buffer. Centrifuge procedure The centrifuge procedure is calling for a centrifuge with a suitable rotor capable of holding 96-well plates (see reference table in catalogue). 1.) Insert the JETWELL 96-well plate into a suitable 2.2 ml waste collection plate (provided with the kit). Make sure, that the JETWELL 96-well plate fits securely. 2.) Add 4 volumes of buffer H1 to 1 volume of PCR assay and mix thoroughly (i.e. mix 400 µl buffer H1 with 100 µl PCR assay). It is not necessary to remove oil or kerosene. Transfer the mix into the wells of the JETWELL 96-well plate. 3.) Centrifuge for 5 min at 300 x g. Check if there is any liquid remaining in one of the wells. If there is any remaining liquid, centrifuge for another 2 min at maximum speed. Discard the flowthrough into the sink and blot the waste collection plate dry on a stack of absorbent paper towels. Re-assemble the JETWELL 96-well plate with the waste collection plate. 4.) Add 900 µl of reconstituted buffer H2 to each well and centrifuge for 2 min at maximum speed. Discard the flowthrough into the sink and blot the waste collection plate dry on a stack of absorbent paper towels. Re-assemble the JETWELL 96-well plate with the waste collection plate and centrifuge for another 5 min at maximum speed to remove the last traces of remaining ethanol. If the wells still smell significantly of ethanol after this centrifugation step, incubate the JETWELL 96-well plate for 10 min at 70°C in an incubator. 5.) Assemble the JETWELL 96-well plate with a 96-well collection plate (provided with the kit), suitable for elution volumes of 80 – 100 µl. Apply 80 µl of 10 mM Tris-Cl (pH 8.5) buffer (or water or TE buffer) directly to the center of each well of the JETWELL 96-well plate. Incubate for 1 min at room temperature, then centrifuge for 2 min with maximum speed. If the recovered volumes per well are uneven after the 2 min centrifugation, centrifuge for another 5 min at maximum speed to collect the remaining eluate. An application volume of 80 µl should yield an eluate volume of approximately 60-62 µl. 6.) Measure the DNA yield spectrophotometrically by measuring the absorptions at 260, 280 and 320 nm, using the 320 nm value as a correction factor. Alternatively, scan a UV spectrum of the sample in the range of 200 – 320 nm. Pure DNA has a A260/A280 ratio of 1.7 – 1.9. JETSTAR 2.0 GIGA FILTER plasmid/cosmid purification protocol (new) Introduction + General Considerations! The handling of the novel JETSTAR 2.0 MEGA/GIGA FILTER system is quite different from the Mini/Midi/Maxi kits, that are based on gravity-flow columns and the clearing of the bacterial lysate by centrifugation. New users are strongly advised to read the entire protocol very carefully before starting the procedure! The JETSTAR 2.0 MEGA/GIGA FILTER system will revolutionize the way of preparing milligram amounts of ultrapure plasmid DNA. This system makes use of the well-established JETSTAR anion-exchange technology, yielding the highest DNA quality available worldwide, combined with a so far never-seen speed in this technology. The JETSTAR 2.0 MEGA prep is for culture volumes of 500 ml - 2.5 litres. The nominal capacity is 2.5 mg of DNA. For high-copy plasmids (i.e. pTZ, pBluescript, pUC and other plasmids that are present in E. coli at concentrations of ³ 4-5 µg/ml) we recommend to use not more than 500 ml of culture. The maximum amount of 2.5 litres should only be used with low-copy plasmids or cosmids. The JETSTAR 2.0 GIGA prep is for culture volumes of 2.5 - 5 litres. The nominal capacity is 10 mg of DNA. For high-copy plasmids (i.e. pTZ, pBluescript, pUC and other plasmids that are present in E. coli at concentrations of ³ 4-5 µg/ml) we recommend to use not more than 2.5 litres of culture. The maximum amount of 5 litres should only be used with low-copy plasmids or cosmids. * Before using the kit for the first time, dissolve the lyophilized RNase (provided with the kit) completely in 1.0 – 1.5 ml of buffer E1. Transfer this concentrated solution back into the buffer E1 bottle and store the reconstituted buffer at 4°C. The final RNase concentration is 100 µg/ml. The RNase-containing buffer E1 is stable for 6 months when stored at 4°C. * Check buffer E2 for SDS precipitation. If SDS is precipitated in buffer E2 due to low transport or storing temperatures, redissolve it by warming in a water bath at 37°C for a few minutes. A new working principle! Although still being based on anion-exchange chromatography, the JETSTAR 2.0 MEGA/GIGA FILTER system is different from the hitherto known Mini/Midi/Maxi columns. The MEGA/GIGA FILTER system is not based on gravity flow-columns, but consists of the anion-exchange resin packed in a vacuum-driven filter cartridge. Additionally the new FILTER system contains an extra filter cartridge for the clarification of the bacterial lysate. Vacuum (-600 to -800 mbar or 20 inch Hg) is applied to both cartridges by a conventional water jet filter pump (being standard equipment in nearly all molecular biology labs) or a membrane pump. What auxiliaries do you need? The JETSTAR 2.0 MEGA/GIGA FILTER system requires the presence of: * A water-jet filter pump (or equivalent vacuum source), capable of generating an underpressure of –600 to –800 mbar (approx. 20 inch Hg), · a 500 ml vacuum-resistant laboratory bottle with 45 mm neck (i.e. DURAN*) for the collection of the cleared lysate, * a 1 litre vacuum-resistant laboratory bottle with 45 mm neck (i.e. DURAN*) for equilibration and the binding and washing steps, * a 100 ml vacuum-resistant laboratory bottle with 45 mm neck (i.e. DURAN*) for the elution step. To avoid the possibility of implosion, do not use plastic/glass bottles or any other vessels that are not designed for the use with vacuum. Do not use plastic/glass bottles or any other vessels that are cracked or scratched. Wear safety glasses when working near a bottle under vacuum. The JETSTAR 2.0 GIGA FILTER Protocol 1.) Setup of FILTER cartridge: Screw the GIGA FILTER cartridge onto a clean 500 ml bottle with 45 mm neck (i.e. DURAN*). Note: Do not overtighten the GIGA FILTER cartridge on the bottle neck, since the GIGA FILTER cartridge plastic may crack. 2.) Harvesting bacterial cells: E. coli cells are pelleted by centrifugation for 3 min at 13,000 x g. Remove all traces of medium carefully. 3.) Cell Resuspension: Add 125 ml of buffer E1 to the pellet and resuspend the cells until the suspension is homogeneous. No cell clumps must be visible. 4.) Cell Lysis: Lyse the bacterial cells by adding 125 ml of buffer E2. Mix gently but thoroughly until a homogeneous lysate is obtained. Due to the release of genomic DNA the mixture is very viscous at this stage. DO NOT VORTEX as this will result in shearing of the genomic DNA! Incubate at room temperature for 5 min. 5.) Neutralization: Neutralize the lysis mix from step 4 with 125 ml of buffer E3. Mix gently but thoroughly until a homogeneous mixture is obtained. DO NOT VORTEX! The liquid of the neutralized lysate must be completely thin-bodied again (not longer viscous). NO remainders of the viscous lysate must be left!!! A white, flocculent precipitate made of proteins, cellular debris, genomic DNA and detergent will form. 6.) Filtration: After mixing with buffer E3 pour the bacterial lysate from step 5 directly into the prepared GIGA FILTER cartridge from step 1. Let stand at room temperature for at least 5 min without agitation!! Then attach a vacuum source to the tubing connector and apply vaccum. Collect the clear flowthrough into the bottle. Keep the vacuum on until all liquid has drained from the unit. Then switch off the vacuum source. Important Note: It is very important to let the lysate stand for at least 5 minutes after the transfer into the cartridge. This is for the precipitate to float and form a layer on top of the lysate. For a quick and convenient filtration it is vital, that the majority of precipitate forms the layer on top of the liquid. If necessary, extend the incubation time of the lysate in the cartridge until the layer has formed. This ensures convenient filtration without clogging. As a rule of thumb, one can expect up to 330 ml filtrate 1-2 min after vacuum has been applied. 7.) Add 50 mls of buffer E5 to the GIGA FILTER cartridge and gently stir the precipitate with a sterile spatula. Connect the vacuum source again and apply vacuum until all liquid has been pulled through completely. The bottle now contains the filtered lysate containing the plasmid DNA. Note: Gentle agitation of the precipitate improves the flow of liquid through the filter unit. 8.) Equilibration: Screw the GIGA cartridge containing the ion-exchange resin onto a 1 litre laboratory bottle with 45 mm neck (i.e. DURAN*) and fill in 200 ml of equilibration buffer E4. Apply vacuum to the cartridge through the side-arm with tubing-connector and suck through the complete amount of liquid. Keep the vacuum on until all liquid has drained from the resin. Discard the flowthrough. 9.) Loading of the lysate: Fill the filtered cleared lysate from step 7 into the cartridge with the equilibrated JETSTAR 2.0 resin and apply vacuum to the cartridge through the side-arm with tubing-connector. Keep the vacuum on until all of the lysate has passed through the resin. You may take an aliquot of the flowthrough for further analysis. 10.) Wash 1: Fill 275 ml of buffer E5 into the cartridge and apply vacuum to the cartridge through the side-arm with tubing-connector. Keep the vacuum on until all liquid has drained from the resin. You may take an aliquot of the flowthrough for further analysis. 11.) Wash 2: Repeat step 10 once with 275 ml of buffer E5. Two successive rounds of washing should be sufficient to remove all impurities (i.e. proteins, degraded RNA, metabolites, dyes) completely. The second wash is particularly necessary when large culture volumes or bacterial strains producing large amounts of contaminants (i.e. RNA, carbohydrates) have been processed. For most preparations the first wash should be sufficient to remove all contaminants completely. 12.) Plasmid Elution: Take off the filter cartridge from the 1 litre bottle and screw it onto a clean, sterile 100 ml laboratory bottle with 45 mm neck (i.e. DURAN*). Pour 100 ml of elution buffer E6 into the cartridge. IMPORTANT NOTE: Apply a soft vacuum (–100 to –200 mbar) to the cartridge through the side-arm with tubing-connector until approximately 30 – 40 ml of buffer E6 have been pulled through the cartridge. Then release the vacuum from the cartridge, so that no further liquid is pulled through the resin. Let stand for 1 min without agitation. Then switch on the vacuum source again and draw the remaining liquid from the resin into the receiver bottle. Keep the vacuum on until all liquid has drained from the resin. You may take an aliquot of the eluate for further analysis. The final DNA yield can be increased by approximately 10% if a second elution step with another 100 ml of buffer E6 is carried out as described. 13.) Plasmid Precipitation: Precipitate the DNA with 0.7 volumes of isopropanol. Spin down the DNA for at least 30 min at ³12,000 x g and 4°C. Discard the supernatant, wash the precipitated DNA with 20 ml of 70-80% ethanol per tube and recentrifuge for 5 min as described. Discard the ethanol and air-dry the DNA pellet completely until all ethanol has evaporated. Redissolve the DNA in a suitable volume of buffer. Plasmid DNA is quite sticky and tends to spread over the whole wall of the centrifuge tube if a fixed angle rotor is used. Therefore we recommend the use of a swing-out rotor that allows centrifugal forces of *12,000 x g (i.e. HB-4 or HB-6 for Sorvall centrifuges). If such a rotor is not available, we suggest the siliconization of the centrifuge tubes with a repellent silane (i.e. dimethyldichlorosilane). JETFLEX Genomic DNA Purification Kit Description of the kit Kit Components / Storage & Stability Additional Materials Required Expected Results Protocols Description of the kit GENOMED’s JETFLEX Genomic DNA Purification Kit provides all reagents necessary to prepare high-quality, high molecular weight genomic DNA from a large variety of samples, among them blood, bone marrow, cell cultures, tissues of animal origin, plant tissues, bacteria (gram-positive and gram-negative) and yeast. After primary lysis of the DNA-containing cells and the nuclei, the unwanted components are removed by a salt precipitation, thus avoiding any handling of organic solvents like phenol or chloroform. The kit contains proteinase K to break up solid samples like a multitude of animal tissues, and RNase A to optionally remove cellular RNA. While residual RNA may not interfere with a variety of subsequent applications, its removal is essential for other ones. Both enzymes are provided with the kit. The purified DNA is an excellent template for PCR amplification, restriction enzyme digestion, Southern blotting, slot blotting and subsequent hybridization. Kit Components / Storage & Stability Component 100 prep Kit (Cat.-No. 600 100) 500 prep Kit (Cat.-No. 600 500) Red Blood Cell (RBC) Lysis Buffer 99 ml 495 ml Cell Lysis Buffer (CLB) 33 ml 165 ml Protein Precipitation Buffer 17 ml 83 ml DNA dissolving buffer 22 ml 110 ml Proteinase K 2 x 26 mg 10 x 26 mg RNase A 22 mg 5 x 22 mg The amounts of buffer given in the table are based on a single prep of 300 µl whole blood or up to 10 mg of solid tissue. Preparation sizes can be varied and scaled up or down proportionally according to the user’s requirements. If the preparation size is scaled up, the number of preparations will obviously decrease proportionally. All components except the enzymes should be stored at room temperature. The enzyme solids should be stored at 4°C. Once dissolved in water, the enzymes should be stored at –20°C to prolong their lifetimes. To avoid repeated freeze-thaw cycles, divide the dissolved enzymes into single-use aliquots. When stored as directed, all components are stable for at least 1 year from the purchase date. Additional Materials Required The following components are not provided with the kit and have to be supplied by the user. --100% isopropanol (2-propanol) --70-80% ethanol --1.5 ml microfuge tubes or 15 ml or 50 ml centrifuge tubes --Pipet and pipet tips --Vortex mixer --Microfuge or clinical centrifuge --Water bath or thermo bloc Expected Results Species & Material Typical DNA yield Proteinase K treatment RNase A treatment Human whole Blood (300 µl) 5 – 15 µg Optional Optional Human whole Blood (2 ml, 10 ml, 50 ml) 50-100 µg, 250-500 µg, 1.25-2.5 mg Optional Optional Cultured Cells 10-30 µg per 3 x 106 cells Optional Required Body Fluids 2-50 µg per ml Optional Optional Solid Animal Tissue 20 – 150 µg per 10 mg Required Required Plant Tissue 10-15 µg per 40 mg Required Required Non-Mammalian Blood 2,5-7,5 mg/ml Optional Required Gram-Negative Bacteria 20 µg per ml Optional Required Gram-Positive Bacteria 10 µg per ml Optional Required Yeast (Gentra, Promega) 5-7 µg per ml No Required Protocols a) Processing of DNA-containing sources / Preparation of nuclear lysates a1) Whole Blood (300 µl, 2 ml, 10 ml, 50 ml) a2) Other body fluids a3) Eucaryotic cell cultures a4) Solid Tissues from animal + plant origin a5) Gram-positive and gram-negative bacteria a6) Yeast b) Preparation of DNA from nuclear lysates c) Re-Purification Protocol for still impure DNA’s a) Processing of DNA-containing sources / Preparation of nuclear lysates a1) Whole Blood Samples (300 µl / 2 ml / 10 ml / 50 ml) a11) Sample Collection and Handling Collect whole blood or buffy coat samples in tubes with a suitable anticoagulant (EDTA, citrate or heparin) to prevent clotting. As EDTA also reduces DNA degradation, GENOMED recommends EDTA for the collection of blood samples. Store fresh blood and buffy coat samples for not longer than 5 days at 4°C for optimal results. If longer storage times are necessary, the samples should be stored frozen at –80°C. Frozen samples are stable at –80°C for at least two years. Before use, blood samples should be thawed quickly in a 37°C water bath and then kept on ice until use. Buffy coat is a concentrate of DNA-containing white blood cells. When processing buffy coat, be careful, not to overload the chemistry of the protocol. 300 µl / 2 ml / 10 ml / 50 ml of whole blood correspond to approximately 15-25 µl / 100-170 µl / 500-850 µl / 2.5-4.25 ml of buffy coat. Adjust the respective amounts of buffy coat with PBS (phosphate-buffered saline, not provided with the kit) to the volumes of the corresponding blood samples and handle them as described in the protocol. a12) Preparation of a DNA-containing lysate from various amounts of whole blood 1.) Add 300 µl / 2 ml / 10 ml / 50 ml of whole blood to a suitable tube with 900 µl / 6 ml / 30 ml / 150 ml of RBC lysis buffer. Mix well by inverting until the mixture is homogeneous and let stand at room temperature for 1 min. If the blood sample is not older than 1 hour after having been drawn from the donor, extend the incubation time from 1 to at least 3 min to ensure complete lysis of the erythrocytes. 2.) Centrifuge for 30 sec. at 13.000 x g (for an initial blood volume of 300 µl) or 10 min at 2.000 x g (for all other volumes). Remove the clear red supernatant, leaving behind a visible white cell pellet. Do not disturb the white cell pellet, which may still appear slightly redish at this stage. It is not necessary to wash the white cell pellet with RBC lysis buffer again as the red stain will be removed during the subsequent procedure. 3.) IMPORTANT: Bump vortex the tube with the white cell pellet vigorously to loosen the cells from the wall. This will greatly enhance cell lysis in the next step. 4.) Add 300 µl / 2 ml / 10 ml / 50 ml of CLB (Cell Lysis Buffer) to the resuspended cells and pipet up and down to lyse the cells. Usually no incubation is required. If cell clumps are visible after mixing, incubate at 37°C or room temperature until the lysate is homogeneous and clear. Samples are stable in CLB for several months at room temperature. CLB will disrupt the cellular structure of the white blood cells and the cell nuclei, thus setting free the genomic DNA. 5.) OPTIONAL: RNase treatment. Add 1.5 µl / 10 µl / 50 µl / 250 µl of RNase solution (4 mg/ml; provided with the kit) to the lysate of step 4 and incubate at 37°C for 10 min. 6.) Go ahead to step b (Preparation of DNA from nuclear lysates). a2) Other body fluids (50 µl, 1 ml) a21) Sample Collection and Handling When body fluid samples are stored at 4°C or on ice, they should be processed quickly. For long-term storage store them at –70 to -80°C. If body fluids with low cell numbers are to be processed, the cells should be concentrated by a centrifugation for 10 min at 2.000 x g. Discard or pipet off the supernatant leaving behind desired volume of residual liquid to thoroughly resuspend the pellet. a22) Preparation of a DNA-containing lysate from various amounts of body fluids 1.) Add 50 µl / 1 ml of body fluid (i.e. plasma, saliva, serum, milk, urine, cerebrospinal fluid, sputum) into a suitable reaction tube containing 250 µl / 5 ml CLB. Mix immediately and thoroughly by pipetting up and down. If the sample has a high protein content, add 550 µl / 11 ml CLB per 50 µl / 1 ml of body fluid. 2.) Incubate at 56°C until the mixture becomes fully clear. OPTIONAL: Add 20 µl / 400 µl proteinase K stock solution (20 mg/ml) and incubate for 1 h to overnight at 56°C until the mixture is fully clear and all cells are lysed. CLB will disrupt the cellular structure of the white blood cells and the cell nuclei, thus setting free the genomic DNA. 3.) OPTIONAL: RNase treatment. Add 10 µl / 200 µl of RNase solution (4 mg/ml; provided with the kit) to the lysate of step 4 and incubate at 37°C for another 10 min. 4.) Go ahead to step b (Preparation of DNA from nuclear lysates). a3) Cultured cells (1-2 x 106 cells / 1-2 x 107 cells) a31) Sample Collection + Handling When preparing DNA from cultured cells, the cells should be placed on ice immediately before processing them. For long-term storage keep the cells at –70 to –80°C. Determine the number of cells using a hemacytometer or other cell counter. a32) Preparation of a DNA-containing lysate from various amounts of cultured cells 1.) Add 1-2 x 106 / 1-2 x 107 cells in balanced salt solution (i.e. PBS, not provided with the kit) into a suitable reaction tube. 2.) Centrifuge for 15 sec. at 13.000 x g (for 1-2 x 106 cells) or 2 min at 2.000 x g (for 1-2 x 107 cells). Remove the supernatant leaving behind desired volume of residual buffer to thoroughly resuspend the cells. 3.) IMPORTANT: Bump vortex the tube with the cell pellet vigorously to loosen the cells from the wall and resuspend them in the residual liquid. This will greatly enhance cell lysis in the next step 4.) Add 300 µl / 3 ml CLB to the resuspended cells and lyse the cells by pipetting up and down. If cell clumps remain visible, incubate at 56°C until the mixture becomes fully clear. OPTIONAL: Add 20 µl / 200 µl proteinase K stock solution (20 mg/ml) and incubate for 1 h to overnight at 56°C until the mixture is fully clear and all cells are lysed. CLB will disrupt the cellular structure of the cultured cells and the cell nuclei, thus setting free the genomic DNA. 5.) OPTIONAL: RNase treatment. Add 10 µl / 100 µl of RNase solution (4 mg/ml; provided with the kit) to the lysate of step 4 and incubate at 37°C for another 10 min. 6.) Go ahead to step b (Preparation of DNA from nuclear lysates). a4) Solid Tissues from animal + plant origin (10 mg / 100 mg) a41) Sample collection and handling Fresh, frozen, fixed or paraffin-embedded tissue samples as well as mouse tails can be processed with the JETFLEX kit. If the tissue samples are to be stored for a longer period of time, they should be stored at –70 to –80°C. Before going into the JETFLEX procedure, tissue samples are best reduced to small pieces (i.e. by grinding in liquid nitrogen with mortar and pestle). When using fresh tissue samples, keep them on ice until cell lysis buffer (CLB) has been added to minimize any degradation of the DNA by the action of DNases. a42) Preparation of a DNA-containing lysate from various amounts of solid tissue 1.) Add up to 10 mg / up to 100 mg fresh tissue or frozen tissue that was ground to small pieces into a suitable reaction tube. Keep tube on ice. When working with plant tissue material, it may be necessary to vary the amount of starting material depending upon species, age, tissue type and genome size. If ever possible, plant tissue samples should be finely ground to disrupt cellular structures before the addition of CLB. 2.) Add 300 µl / 3 ml CLB to the tissue sample. If the tissue sample wasn’t reduced to small pieces, homogenize the sample thoroughly using 30-50 strokes with a suitable tube pestle. Add 20 µl / 200 µl proteinase K stock solution (20 mg/ml) and incubate for 1 h to overnight at 56°C until the mixture is fully clear and the tissue structure is fully desintegrated. CLB will disrupt the cellular structure of the tissue cells and the nuclei, thus setting free the genomic DNA. 3.) OPTIONAL: RNase treatment. Add 10 µl / 100 µl of RNase solution (4 mg/ml; provided with the kit) to the lysate of step 4 and incubate at 37°C for another 10 min. 4.) Go ahead to step b (Preparation of DNA from nuclear lysates). a5) Gram-positive and Gram-negative bacteria (500 µl / 5 ml) a51) Preparation of a DNA-containing lysate from various amounts of bacterial culture 1.) Add 500 µl / 5 ml of bacterial culture (i.e. after overnight growth) into a suitable reaction tube. 2.) Centrifuge for 1 min. at 13.000 x g to pellet the bacterial cells. Remove the supernatant quantitatively with a pipet. For gram-positive bacteria proceed with step 3, for gram-negative bacteria go directly to step 4. 3.) If gram-positive bacteria are to be processed: Due to the high murein content of the cell walls of gram-positive bacteria, a much rougher treatment is necessary to disrupt the cells. Therefore resuspend the cells in 300 µl / 3 ml of a buffer containing 50 mM Tris-Cl (pH 8,0) / 10 mM EDTA with 5 mg/ml lysozyme (not provided with the kit). After having obtained a homogeneous suspension incubate for 30 min at 37°C. Then centrifuge for 1 min at 13.000 x g / 5 min at 2000 x g to pellet the cells again. Remove the supernatant. For most gram-positive species a treatment with lysozyme is sufficient to weaken the cell wall. However, some Staphylococcus species require an additional presence of 1 mg/ml lysostaphin (not provided with the kit) to get the cell walls efficiently lysed. 4.) Add 300 µl / 3 ml CLB to the resuspended cells and lyse the cells by pipetting up and down. If cell clumps remain visible, incubate at 56°C until the mixture becomes fully clear. OPTIONAL: Add 10 µl / 100 µl proteinase K stock solution (20 mg/ml) and incubate for 1 h to overnight at 56°C until the mixture is fully clear and all cells are lysed. CLB will disrupt the cellular structure of the bacterial cells, thus setting free the genomic DNA. 5.) OPTIONAL: RNase treatment. Add 10 µl / 100 µl of RNase solution (4 mg/ml; provided with the kit) to the lysate of step 4 and incubate at 37°C for another 10 min. 6.) Go ahead to step b (Preparation of DNA from nuclear lysates). a6) Yeast (1 ml / 10 ml) a61) Preparation of a DNA-containing lysate from various amounts of yeast culture 1.) Grow the Saccharomyces culture to saturation in YPD or YEPD (= 1-2 x 108 cells/ml). Harvest 1 ml / 10 ml of the cells by centrifugation (1 ml: 13.000 x g for 1 min, 10 ml: 2.000 x g for 3 min) and remove the supernatant quantitatively. 2.) Wash the cells by resuspending them in 300µl / 3 ml buffer YS1 (0.9 M sorbitol / 0.1 M Na2EDTA [pH 7.5] / 14 mM 2-mercaptoethanol, not provided with the kit).This is for the removal of remaining media components and to ensure optimal enzymatic performance with the Zymolyase in step 4. 3.) Spin down the cells at 13.000 x g for 1 min and discard the supernatant. Resuspend the cells in 300 µl / 3 ml buffer YS1. The cell suspension must be homogeneous; no cell clumps must be visible! 4.) Add 30 µl / 300 µl of Zymolyase 60,000 (Miles; 2 mg/ml) and incubate at 37°C for 20 - 30 min. Zymolyase will break down the cell wall of the yeast cells enzymatically during incubation. Equivalent enzymes to Zymolyase are Lyticase (Sigma, Cat.-No. L8137) or Zymolase (ICI, Cat.-No. 32-093-2). These enzymes should be diluted from their respective stock solutions in distilled water to a final concentration of 1000 U/ml and the incubation with them be performed for at least 30 min at 30°C. Stock solutions of these enzymes should be stored in aliquots at -20°C and each aliquot only used once. Monitor spheroplast formation by examination to detergent sensitivity: a small sample of cells is diluted into 1% SDS, and spheroplasting is sufficient when greater than 90% of the cells burst when examined under the microscope. 5.) Spin down the spheroplasts at 13.000 x g for 1 min (for an initial volume of 1 ml yeast culture) or 5000 x g for 10 min (for an initial volume of 10 ml yeast culture). Remove supernatant. 6.) Add 300 µl / 3 ml CLB to the yeast cells and lyse them by pipetting up and down. If cell clumps remain visible, incubate at 56°C until the mixture becomes fully clear. CLB will disrupt the structures of the spheroblasts, thus setting free the genomic DNA. 7.) OPTIONAL: RNase treatment. Add 10 µl / 100 µl of RNase solution (4 mg/ml; provided with the kit) to the lysate of step 4 and incubate at 37°C for another 10 min. 8.) Go ahead to step b (Preparation of DNA from nuclear lysates). b) Preparation of DNA from nuclear lysates 1.) Before processing any cellular lysate any further, make sure that it has reached room temperature again. 2.) For each 300 µl / 3 ml of cellular lysate from one of the introductory procedures above add 150 µl / 1.5 ml of protein precipitation buffer. Mix thoroughly by vortexing for 20 seconds to obtain a homogeneous suspension. 3.) Centrifuge at 13.000 – 16.000 x g for 3 min at room temperature. The precipitated proteins should form a compact pellet. The supernatant should be clear. If the resultant pellet is small or loose, showing the tendency to be co-transfered with the supernatant, vortex again and chill the mixture on ice for 5 min. Then centrifuge again for 3 min at 13.000 – 16.000 x g either at room temperature or in a chilled centrifuge at 4°C. 4.) Transfer the clear supernatant into a fresh tube. Add 1 vol. of isopropanol (2-propanol). 5.) Mix thoroughly by multiple inverting until a homogeneous mixture is obtained. Avoid vortexing. 6.) Centrifuge at 13.000 – 16.000 x g for 3 min at room temperature. The precipitated DNA should be visible as a small white pellet. 7.) Decant the supernatant and let residual liquid drain on a sheet of absorbant paper towel for a few minutes. Add 1 ml / 10 ml of 70-80% ethanol to the tube and wash the DNA pellet by inverting several times. 8.) Centrifuge at 13.000 – 16.000 x g for 1 min. Carefully decant the ethanolic supernatant. The DNA pellet can sit very loosely at the wall of the tube at this stage. Be very careful not to discard your DNA. 9.) Let residual liquid drain on a sheet of absorbant paper towel for a few minutes. Evaporate residual ethanol by a 10 min incubation at elevated temperature (50-56°C). One can also allow the DNA pellet to air-dry at room temperature, but it may take significantly longer for all remaining ethanol to fully evaporate. 10.) Add a suitable volume of DNA Dissolving Buffer to the DNA pellet. Dissolve the DNA by standing at room temperature overnight. The dissolution process can be enhanced by incubating at 65°C for up to 1 h. 11.) Store the DNA at 4°C. The dissolved DNA should be quantified spectrophotometrically. Pure DNA has a A260/A280 ratio of 1.7 – 1.9. One OD260 corresponds to a DNA concentration of 50 µg/ml. Analysis on an analytical agarose gel should reveal the majority of DNA migrating at a size of ³ 50 – 150 kb. c) Re-Purification Protocol for still impure DNA’s Pure DNA should dissolve readily in the provided DNA Dissolving Buffer, giving a clear, colorless solution. If the DNA solution still contains insoluble matter or is stained in some way, or the A260/A280 ratio is <1,70, it is still contaminated with protein or another impurity. This may occur, when i.e. the chemistry of the purification procedure has been overloaded. Still contaminated samples may be further purified easily using the following protocol. 1.) For every 100 µl of contaminated DNA solution add 500 µl of CLB and mix thoroughly by inverting or pipetting up and down. If necessary, incubate at 37°C until all particulate matter has dissolved. IMPORTANT: Cool the sample down to room temperature before proceeding to step 2. 2.) For every 600 µl of DNA solution/CLB mixture add 300 µl of protein precipitation buffer and vortex vigorously for 20 sec to obtain a homogeneous mixture. 3.) Centrifuge for 3 min at 13.000 – 16.000 x g (or 10 min at 5.000 x g) to pellet the precipitated impurities. If a pellet is not visible, re-vortex and incubate the sample for 5 min on ice. Then re-centrifuge the sample as described. 4.) Transfer the supernatant into a clean suitable receptacle without transferring the precipitated impurities. 5.) Add 1 vol. of isopropanol (2-propanol) to the transferred supernatant and mix thoroughly by multiple inverting. The precipitated DNA should become visible as a thread-like matter. 6.) Centrifuge for 1-3 min at 12.000 x g to collect the precipitated DNA at the bottom of the tube. Discard the supernatant and let residual liquid drain from the DNA pellet by placing the tube upside-down on a sheet of absorbant paper towel. 7.) Wash the DNA pellet with a suitable volume of 70-80% ethanol. Centrifuge again for 1-3 min at 12.000 x g to collect the DNA at the bottom of the tube. Discard the ethanolic supernatant. The DNA pellet may sit very loose at the tube’s wall at this stage. Be careful not to discard the DNA. 8.) Let residual liquid drain on a sheet of absorbant paper towel for a few minutes. Evaporate residual ethanol by a 10 min incubation at elevated temperature (50-56°C). One can also allow the DNA pellet to air-dry at room temperature, but it may take significantly longer for all remaining ethanol to fully evaporate. 9.) Add a suitable volume of DNA Dissolving Buffer to the DNA pellet. Dissolve the DNA by standing at room temperature overnight. The dissolution process can be enhanced by incubating at 65°C for up to 1 h. 10.) Store the DNA at 4°C. The dissolved DNA should be quantified spectrophotometrically. Pure DNA has a A260/A280 ratio of 1.7 – 1.9. One OD260 corresponds to a DNA concentration of 50 µg/ml. Analysis on an analytical agarose gel should reveal the majority of DNA migrating at a size of ³ 50 – 150 kb. JETSTAR 2.0 MEGA FILTER plasmid/cosmid purification protocol (new) Introduction + General Considerations! The handling of the novel JETSTAR 2.0 MEGA/GIGA FILTER system is quite different from the Mini/Midi/Maxi kits, that are based on gravity-flow columns and the clearing of the bacterial lysate by centrifugation. New users are strongly advised to read the entire protocol very carefully before starting the procedure! The JETSTAR 2.0 MEGA/GIGA FILTER system will revolutionize the way of preparing milligram amounts of ultrapure plasmid DNA. This system makes use of the well-established JETSTAR anion-exchange technology, yielding the highest DNA quality available worldwide, combined with a so far never-seen speed in this technology. The JETSTAR 2.0 MEGA prep is for culture volumes of 500 ml - 2.5 litres. The nominal capacity is 2.5 mg of DNA. For high-copy plasmids (i.e. pTZ, pBluescript, pUC and other plasmids that are present in E. coli at concentrations of ³ 4-5 µg/ml) we recommend to use not more than 500 ml of culture. The maximum amount of 2.5 litres should only be used with low-copy plasmids or cosmids. The JETSTAR 2.0 GIGA prep is for culture volumes of 2.5 - 5 litres. The nominal capacity is 10 mg of DNA. For high-copy plasmids (i.e. pTZ, pBluescript, pUC and other plasmids that are present in E. coli at concentrations of ³ 4-5 µg/ml) we recommend to use not more than 2.5 litres of culture. The maximum amount of 5 litres should only be used with low-copy plasmids or cosmids. * Before using the kit for the first time, dissolve the lyophilized RNase (provided with the kit) completely in 1.0 – 1.5 ml of buffer E1. Transfer this concentrated solution back into the buffer E1 bottle and store the reconstituted buffer at 4°C. The final RNase concentration is 100 µg/ml. The RNase-containing buffer E1 is stable for 6 months when stored at 4°C. * Check buffer E2 for SDS precipitation. If SDS is precipitated in buffer E2 due to low transport or storing temperatures, redissolve it by warming in a water bath at 37°C for a few minutes. A new working principle! Although still being based on anion-exchange chromatography, the JETSTAR 2.0 MEGA/GIGA FILTER system is different from the hitherto known Mini/Midi/Maxi columns. The MEGA/GIGA FILTER system is not based on gravity flow-columns, but consists of the anion-exchange resin packed in a vacuum-driven filter cartridge. Additionally the new FILTER system contains an extra filter cartridge for the clarification of the bacterial lysate. Vacuum (-600 to -800 mbar or 20 inch Hg) is applied to both cartridges by a conventional water jet filter pump (being standard equipment in nearly all molecular biology labs) or a membrane pump. What auxiliaries do you need? The JETSTAR 2.0 MEGA/GIGA FILTER system requires the presence of: * A water-jet filter pump (or equivalent vacuum source), capable of generating an underpressure of –600 to –800 mbar (approx. 20 inch Hg), · a 200 – 500 ml vacuum-resistant laboratory bottle with 45 mm neck (i.e. DURAN*) for the collection of the cleared lysate, * a 1 litre vacuum-resistant laboratory bottle with 45 mm neck (i.e. DURAN*) for equilibration and the binding and washing steps, * a 100 ml vacuum-resistant laboratory bottle with 45 mm neck (i.e. DURAN*) for the elution step. To avoid the possibility of implosion, do not use plastic/glass bottles or any other vessels that are not designed for the use with vacuum. Do not use plastic/glass bottles or any other vessels that are cracked or scratched. Wear safety glasses when working near a bottle under vacuum. The JETSTAR 2.0 MEGA FILTER Protocol 1.) Setup of FILTER cartridge: Screw the MEGA FILTER cartridge onto a clean 200 ml or 500 ml bottle with 45 mm neck (i.e. DURAN*). Note: Do not overtighten the MEGA FILTER cartridge on the bottle neck, since the MEGA FILTER cartridge plastic may crack. 2.) Harvesting bacterial cells: E. coli cells are pelleted by centrifugation for 3 min at 13,000 x g. Remove all traces of medium carefully. 3.) Cell Resuspension: Add 50 ml of buffer E1 to the pellet and resuspend the cells until the suspension is homogeneous. No cell clumps must be visible. 4.) Cell Lysis: Lyse the bacterial cells by adding 50 ml of buffer E2. Mix gently but thoroughly until a homogeneous lysate is obtained. Due to the release of genomic DNA the mixture is very viscous at this stage. DO NOT VORTEX as this will result in shearing of the genomic DNA! Incubate at room temperature for 5 min. 5.) Neutralization: Neutralize the lysis mix from step 4 with 50 ml of buffer E3. Mix gently but thoroughly until a homogeneous mixture is obtained. DO NOT VORTEX! The liquid of the neutralized lysate must be completely thin-bodied again (not longer viscous). NO remainders of the viscous lysate must be left!!! A white, flocculent precipitate made of proteins, cellular debris, genomic DNA and detergent will form. 6.) Filtration: After mixing with buffer E3 pour the bacterial lysate from step 5 directly into the prepared MEGA FILTER cartridge from step 1. Let stand at room temperature for at least 5 min without agitation!! Then attach a vacuum source to the tubing connector and apply vaccum. Collect the clear flowthrough into the bottle. Keep the vacuum on until all liquid has drained from the unit. Then switch off the vacuum source. Important Note: It is very important to let the lysate stand for at least 5 minutes after the transfer into the cartridge. This is for the precipitate to float and form a layer on top of the lysate. For a quick and convenient filtration it is vital, that the majority of precipitate forms the layer on top of the liquid. If necessary, extend the incubation time of the lysate in the cartridge until the layer has formed. This ensures convenient filtration without clogging. As a rule of thumb, one can expect up to 125 ml filtrate 1-2 min after vacuum has been applied. 7.) Add 50 mls of buffer E5 to the MEGA FILTER cartridge and gently stir the precipitate with a sterile spatula. Connect the vacuum source again and apply vacuum until all liquid has been pulled through completely. The bottle now contains the filtered lysate containing the plasmid DNA. Note: Gentle agitation of the precipitate improves the flow of liquid through the filter unit. 8.) Equilibration: Screw the MEGA cartridge containing the ion-exchange resin onto a 1 litre laboratory bottle with 45 mm neck (i.e. DURAN*) and fill in 100 ml of equilibration buffer E4. Apply vacuum to the cartridge through the side-arm with tubing-connector and suck through the complete amount of liquid. Keep the vacuum on until all liquid has drained from the resin. Discard the flowthrough. 9.) Loading of the lysate: Fill the filtered cleared lysate from step 7 into the cartridge with the equilibrated JETSTAR 2.0 resin and apply vacuum to the cartridge through the side-arm with tubing-connector. Keep the vacuum on until all of the lysate has passed through the resin. You may take an aliquot of the flowthrough for further analysis. 10.) Wash 1: Fill 175 ml of buffer E5 into the cartridge and apply vacuum to the cartridge through the side-arm with tubing-connector. Keep the vacuum on until all liquid has drained from the resin. You may take an aliquot of the flowthrough for further analysis. 11.) Wash 2: Repeat step 10 once with 175 ml of buffer E5. Two successive rounds of washing should be sufficient to remove all impurities (i.e. proteins, degraded RNA, metabolites, dyes) completely. The second wash is particularly necessary when large culture volumes or bacterial strains producing large amounts of contaminants (i.e. RNA, carbohydrates) have been processed. For most preparations the first wash should be sufficient to remove all contaminants completely. 12.) Plasmid Elution: Take off the filter cartridge from the 1 litre bottle and screw it onto a clean, sterile 100 ml laboratory bottle with 45 mm neck (i.e. DURAN*). Pour 50 ml of elution buffer E6 into the cartridge. IMPORTANT NOTE: Apply a soft vacuum (–100 to –200 mbar) to the cartridge through the side-arm with tubing-connector until approximately 5 – 10 ml of buffer E6 have been pulled through the cartridge. Then release the vacuum from the cartridge, so that no further liquid is pulled through the resin. Let stand for 1 min without agitation. Then switch on the vacuum source again and draw the remaining liquid from the resin into the receiver bottle. Keep the vacuum on until all liquid has drained from the resin. You may take an aliquot of the eluate for further analysis. The final DNA yield can be increased by approximately 10% if a second elution step with another 50 ml of buffer E6 is carried out as described. 13.) Plasmid Precipitation: Precipitate the DNA with 0.7 volumes of isopropanol. Spin down the DNA for at least 30 min at ³12,000 x g and 4°C. Discard the supernatant, wash the precipitated DNA with 20 ml of 70-80% ethanol per tube and recentrifuge for 5 min as described. Discard the ethanol and air-dry the DNA pellet completely until all ethanol has evaporated. Redissolve the DNA in a suitable volume of buffer. Plasmid DNA is quite sticky and tends to spread over the whole wall of the centrifuge tube if a fixed angle rotor is used. Therefore we recommend the use of a swing-out rotor that allows centrifugal forces of *12,000 x g (i.e. HB-4 or HB-6 for Sorvall centrifuges). If such a rotor is not available, we suggest the siliconization of the centrifuge tubes with a repellent silane (i.e. dimethyldichlorosilane). JETWELL 96-well Blood Genomic DNA Purification system The JETWELL 96-well blood genomic DNA purification system can be used to prepare genomic DNA from whole blood, buffy coat, plasma, serum or other body fluids, as well as lymphocytes, bone marrow or cultured cells. For the long-term storage of proteinase K and RNase A we recommend dissolving the enzymes in water as stated on the accompanying label, divide it into single-use aliquots and store them at –20°C. Repeated freezing and thawing should be avoided. Introduction The JETWELL 96-well blood genomic DNA purification system is for the simultaneous purification of DNA from 96 samples originating from a wide range of clinical starting materials. Sample types include fresh or frozen whole blood (treated with either EDTA, citrate or heparine as an anticoagulant), plasma, serum, bone marrow, other body fluids, lymphocytes and cultured cells that have a normal set of chromosomes. The kit combines the selective binding properties of a unique silica membrane with a convenient high-throughput 96-well format. The DNA is highly pure and suitable for all enzymatic in vitro applications, like PCR, Southern blotting or restriction enzyme digestion. The DNA isolated with the kit has an average size of 30 – 50 kb, making it an ideal template for PCR amplification. DNA of this length denatures completely during thermal cycling and is amplified very efficiently. The kit doesn’t use any toxic reagents like phenol, chloroform or ethidium bromide and yields DNA that is directly eluted in Tris or another low-salt buffer, thus being ready-to-use. All components should be at room temperature when used. The Principle The procedure is designed for 200 – 400 µl blood samples. Samples being smaller than 200 µl in size should be adjusted to 200 µl with PBS before starting the procedure. For sample volumes larger than 200 µl, the amount of lysis buffer K1 and the other reagents added to the sample before loading must be increased proportionally. The DNA-containing blood cells are lysed with proteinase K in the presence of a chaotropic salt. After lysis has occured, absolute ethanol is added to ensure optimal binding to the membrane. This mixture is further processed over the silica membrane in the 96-well plate. The JETWELL 96-well plate procedure The blood sample treated with lysis buffer, proteinase K and ethanol is loaded into the particular wells of the 96-well plate and processed over the silica membrane at the bottom of each well by centrifugation. During the processing over the membrane the DNA will bind reversibly to the silica. After the removal of the unwanted components (i.e. proteins, hemin, red blood stain, salts) during the following washing steps, the DNA is eluted in a low-salt buffer (10 mM Tris-Cl [pH 9.0], provided). The isolated DNA can directly be used for downstream applications. When carrying out the procedure without the use of RNase, DNA and RNA will be purified simultaneously. RNA may inhibit some downstream enzymatic reactions. So, if RNA-free genomic DNA is required, the addition of 10 µl RNase A (20 mg/ml) per 200 µl of whole blood during the procedure is recommended (see protocol). Protocol The current protocol is for a centrifuge-driven 96-well procedure: Preliminary steps 1.) Reconstitute buffers KX and K2 with absolute ethanol as described on the bottle’s label. After having added the ethanol to the buffer concentrate, mix well by shaking. 2.) Be careful when handling buffers K1 and KX as these buffers contain guanidine hydrochloride. This substance is an irritant. Wear gloves and goggles when handling these buffers. 3.) If a precipitate is present in buffer K1, re-dissolve it by a short incubation at 50 – 55°C. 4.) All pipetting steps should be carried out with a suitable multichannel pipette. 5.) All steps, where vigorous mixing is required should be carried out by sealing the respective plate tightly with a suitable plastic mat (provided with the kit) and shaking vigorously up and down for at least 15 seconds, thereby holding the block with both hands. Shaking the block with only one hand will result in less efficient mixing in the wells on the side of the block held in the hand, with lower yields being obtained from those wells. Centrifuge procedure The centrifuge procedure is calling for a centrifuge with a suitable rotor capable of holding 96-well plates (see reference table in catalogue). During all centrifugation steps (except the elution step) the openings of the JETWELL 96-well plate should be sealed with adhesive air-permeative tape to prevent cross-contamination. Air permeability is very important to avoid underpressure in the wells during centrifugation. An adhesive tape should also be used when re-using plates containing unused wells. Label used wells of a JETWELL 96-well plate with a waterproof marker pen. Cover unused wells with tape and store the JETWELL 96-well plate in the bag in which it was supplied. Before starting the next run, remove the tape and cover the previously used wells with fresh tape. 1.) Insert the JETWELL 96-well plate into the waste collection plate. Make sure, that the JETWELL 96-well plate fits securely into the waste collection plate. 2.) Pipet 20 µl of proteinase K solution (20 mg/ml) into the bottom of the wells of a 2 ml Deep Well block. Make sure, that all pipet tips touch near the base of each well and the rims of the wells remain dry. Per well add 200 µl of whole blood, plasma, serum or other body fluids, or 5 x 106 – 1 x 107 lymphocytes or cultured cells in 200 µl PBS to the pre-dispensed proteinase K. Add 200 µl of buffer K1 to each sample, taking care not to wet the rims of the wells. Seal the plate with the plastic mat provided and mix thoroughly by shaking vigorously for at least 15 seconds. IMPORTANT NOTE: If the sample contains both DNA and RNA, both types of nucleic acids will be purified. If RNA-free genomic DNA is to be prepared, add 10 µl of the RNAse stock solution (20 mg/ml; provided) to each sample before buffer K1 is added. For efficient lysis, it is essential that the samples and buffer K1 are mixed thoroughly to yield a homogeneous mixture. Hold the block with both hands and shake up and down vigorously. 3.) Centrifuge briefly in a 96-well plate rotor at 3,000 rpm to collect all liquid at the bottom of each well. Allow centrifuge to reach 3,000 rpm, then stop the centrifuge. 4.) Incubate the block with the blood samples for 10 min at 70°C either in an incubator or a water bath. Placing a weight on top of the round-well block will prevent the lids popping off during incubation. 5.) Centrifuge briefly in a 96-well plate rotor at 3,000 rpm to collect all liquid at the bottom of each well. Allow centrifuge to reach 3,000 rpm, then stop the centrifuge. 6.) Remove the plastic mat and add 200 µl of absolute ethanol (96-100%) to each well. If the sample volume is greater than 200 µl, increase the amount of ethanol proportionally, i.e. when processing a 400 µl blood sample add 400 µl of ethanol. 7.) Seal the well swith the plastic mat again and shake vigorously for at least 15 seconds again. 8.) Centrifuge briefly in a 96-well plate rotor at 3,000 rpm to collect all liquid at the bottom of each well. Allow centrifuge to reach 3,000 rpm, then stop the centrifuge. 9.) Carefully apply the mixture from step 9 to the JETWELL 96-well plate on the waste collection plate from step 1. Take care not to wet the rims of the wells to avoid aerosol formation during centrifugation. 10.) Centrifuge for 2 min at maximum speed. Check if there is any liquid remaining in one of the wells. If there is any remaining liquid, centrifuge for another 2 min at maximum speed. Discard the flowthrough into the sink and blot the waste collection plate dry on a stack of absorbent paper towels. Re-assemble the JETWELL 96-well plate with the waste collection plate. 11.) Add 500 µl of reconstituted buffer KX to each well and centrifuge for 2 min at maximum speed. Discard the flowthrough into the sink and blot the waste collection plate dry on a stack of absorbent paper towels. Re-assemble the JETWELL 96-well plate with the waste collection plate. 12.) Perform another washing step with 900 µl of reconstituted buffer K2. After having discarded the flowthrough and reassembled the JETWELL 96-well plate with the waste collection plate, centrifuge for another 5 min at maximum speed to remove the last traces of remaining ethanol. If the wells still smell significantly of ethanol after this centrifugation step, incubate the JETWELL 96-well plate for 10 min at 70°C in an incubator. 13.) Assemble the JETWELL 96-well plate with a 96-well collection plate, suitable for elution volumes of 80 – 100 µl. Apply 200 µl of 10 mM Tris-Cl (pH 8.5) buffer (or water or TE buffer) prewarmed to 70°C directly to the center of each well of the JETWELL 96-well plate. Incubate for 5 min at room temperature, then centrifuge for 2 min with maximum speed. If the recovered volumes per well are uneven after the 2 min centrifugation, centrifuge for another 5 min at maximum speed to collect the remaining eluate. 14.) Measure the DNA yield spectrophotometrically by measuring the absorptions at 260, 280 and 320 nm, using the 320 nm value as a correction factor. Alternatively, scan a UV spectrum of the sample in the range of 200 – 320 nm. Pure DNA has a A260/A280 ratio of 1.7 – 1.9. JETWELL 96-well General DNA CleanUp system Introduction The JETWELL 96-well General DNA CleanUp system is for the simultaneous purification of 96 in vitro DNA assays, i.e. from enzymatic treatments with restriction enzymes or polymerases or from crude plasmid preparations. Each well has a DNA capacity of ³20 µg. The DNA is highly pure and suitable for all enzymatic in vitro applications, like fluorescent sequencing, radioactive sequencing, restriction enzyme digestion, cloning procedures and microarray analysis. The kit doesn’t use any toxic reagents like phenol, chloroform or ethidium bromide and yields DNA that is directly eluted in Tris or another low-salt buffer, thus being ready-to-use. All components should be at room temperature when used. The Principle The JETWELL 96-well General DNA CleanUp system combines the convenience of multiwell technology with the selective binding properties of a proprietary silica membrane. The binding conditions provided with the kit are optimized for the efficient recovery of DNAs ranging in size from 80 bp to 20 kb. Restriction enzymes, DNA polymerases, other DNA-modifying enzymes, degraded RNA, oligomers with a length of up to 40 nucleotides, unincorporated dNTP’s, and salts are removed to an extent of ³99.5%. The JETWELL 96-well plate procedure The DNA to be purified is mixed with binding buffer and loaded into the particular wells of the 96-well plate. The mixture is then processed over the silica membrane at the bottom of each well by centrifugation. During the processing over the membrane the DNA will bind reversibly to the silica. After the removal of the unwanted components (i.e. enzymes, bacterial proteins and metabolites, oligos, residual degraded RNA, dNTP’s, salts) during the following washing step, the DNA is eluted in a low-salt buffer (10 mM Tris-Cl [pH 9.0], provided). The isolated DNA can directly be used for downstream applications. Protocol The current protocol is for a centrifuge-driven 96-well procedure: Preliminary steps 1.) Reconstitute buffer M2 with absolute ethanol as described on the bottle’s label. 2.) Be careful when handling buffer M1 as this buffer contains guanidine hydrochloride. This substance is an irritant. Wear gloves and goggles when handling this buffer. Centrifuge procedure The centrifuge procedure is calling for a centrifuge with a suitable rotor capable of holding 96-well plates (see reference table in catalogue). 1.) Insert the JETWELL 96-well plate into a suitable 2.2 ml waste collection plate (provided with the kit). Make sure, that the JETWELL 96-well plate fits securely. 2.) Add 4 volumes of buffer M1 to 1 volume of DNA solution and mix thoroughly (i.e. mix 400 µl buffer M1 with 100 µl DNA solution). Transfer the mixes into the wells of the JETWELL 96-well plate. 3.) Centrifuge for 5 min at 300 x g. Check if there is any liquid remaining in one of the wells. If there is any remaining liquid, centrifuge for another 2 min at maximum speed. Discard the flowthrough into the sink and blot the waste collection plate dry on a stack of absorbent paper towels. Re-assemble the JETWELL 96-well plate with the waste collection plate. 4.) Add 900 µl of reconstituted buffer M2 to each well and centrifuge for 2 min at maximum speed. Discard the flowthrough into the sink and blot the waste collection plate dry on a stack of absorbent paper towels. Re-assemble the JETWELL 96-well plate with the waste collection plate and centrifuge for another 5 min at maximum speed to remove the last traces of remaining ethanol. If the wells still smell significantly of ethanol after this centrifugation step, incubate the JETWELL 96-well plate for 10 min at 70°C in an incubator. 5.) Assemble the JETWELL 96-well plate with a 96-well collection plate (provided with the kit), suitable for elution volumes of 80 – 100 µl. Apply 80 µl of 10 mM Tris-Cl (pH 8.5) buffer (or water or TE buffer) directly to the center of each well of the JETWELL 96-well plate. Incubate for 1 min at room temperature, then centrifuge for 2 min with maximum speed. If the recovered volumes per well are uneven after the 2 min centrifugation, centrifuge for another 5 min at maximum speed to collect the remaining eluate. An application volume of 80 µl should yield an eluate volume of approximately 60-62 µl. 6.) Measure the DNA yield spectrophotometrically by measuring the absorptions at 260, 280 and 320 nm, using the 320 nm value as a correction factor. Alternatively, scan a UV spectrum of the sample in the range of 200 – 320 nm. Pure DNA has a A260/A280 ratio of 1.7 – 1.9.  
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