| Description | The Succinic Acid (Succinate) assay kit is suitable for the specific assay of succinic acid in wine, cheese, eggs, sauce and other food products. Succinic acid (or succinate) is found in all plant and animal materials as a result of the central metabolic role played by this dicarboxylic acid in the The Succinic Acid (Succinate) assay kit is suitable for the specific assay of succinic acid in wine, cheese, eggs, sauce and other food products. Succinic acid (or succinate) is found in all plant and animal materials as a result of the central metabolic role played by this dicarboxylic acid in the Citric Acid Cycle. Succinic acid concentrations are monitored in the manufacture of numerous foodstuffs and beverages, including wine, soy sauce, soy bean flour, fruit juice and dairy products (e.g. cheese).Product Description： Succinic acid is found in all plant and animal materials as a result of the central metabolic role played by this dicarboxylic acid in the Citric Acid Cycle. Succinic acid concentrations are monitored in the manufacture of numerous foodstuffs and beverages, including wine, soy sauce, soy bean flour, fruit juice and dairy products (e.g. cheese). The ripening process of apples can be followed by monitoring the falling levels of succinic acid. The occurrence of > 5 mg/kg of this acid in egg and egg products is indicative of microbial contamination. Apart from use as a flavouring agent in the food and beverage industries, succinic acid finds many other non-food applications, such as in the production of dyes, drugs, perfumes, lacquers, photographic chemicals and coolants. Preparation Instructions：Suitable for succinate determination in food, beverage, agricultural products, and other biological samples.Note for Content：The number of manual tests per kit can be doubled if all volumes are halved. This can be readily accommodated using the MegaQuantTM Wave Spectrophotometer (D-MQWAVE).Browse all of our organic acid assay kits.Principle：The Succinate Assay Kit provides a simple, one step assay for measuring succinate. In this assay succinate is converted to pyruvate which reacts with specific reagents and dye to form a colored product. The color intensity at 570 nm or fluorescencAdvantages：Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.Very competitive price (cost per test)All reagents stable for > 2 years as suppliedVery rapid reaction (even at room temperature)Mega-Calc™ software tool is available from our website for hassle-free raw data processingStandard includedSuitable for manual, microplate and auto-analyser formats... Read More | Products contentN665730Component24 T96 TStorageN665730ATPS V50 144 µL576 µL-20℃. Avoid freeze/thaw cycle.N665730B5×FA Reaction Buffer144 µL576 µL-20℃. Avoid freeze/thaw cycle.N665730C2×HiFidelity PCR Mix600 µL2×1.2 mL-20℃. Avoid freeze/thaw Products contentN665730Component24 T96 TStorageN665730ATPS V50 144 µL576 µL-20℃. Avoid freeze/thaw cycle.N665730B5×FA Reaction Buffer144 µL576 µL-20℃. Avoid freeze/thaw cycle.N665730C2×HiFidelity PCR Mix600 µL2×1.2 mL-20℃. Avoid freeze/thaw cycle.N665730DPPM48 µL192 µL-20℃. Avoid freeze/thaw cycle.* This kit is suitable for human genomic DNA library construction with a starting template DNA input of 50 ng. We also have transposase library construction kits for human genomic DNA starting at 5 ng and 1 ng, so it is recommended to use different kits for different starting amounts of DNA in order to obtain higher quality libraries. Products IntroductionThis kit is developed for Illumina's high-throughput sequencing platform and provides the enzyme premix system and reaction buffer for genomic DNA library construction, including all components except PCR primers. Compared with the traditional library construction kits, this kit adopts the new transposase method for library construction, which can complete DNA fragmentation, end repair and junction reaction in one simple enzymatic reaction, significantly reducing the amount of template, reducing the number of experimental steps, and shortening the time of library construction; it adopts the high-fidelity DNA polymerase for library enrichment, and the preference-free PCR amplification can expand the coverage area of the sequence, which can be used for efficient and effective sequencing. The use of high-fidelity DNA polymerase for library enrichment and preference-free PCR amplification broadens the coverage area of the sequence and enables efficient preparation of DNA libraries for Illumina's second-generation sequencing platform. The kit is suitable for DNA libraries with a starting template of 50 ng, and all reagents in the kit have been subjected to strict quality control and functional validation to maximize the stability and reproducibility of library construction. Product Features ● DNA fragmentation and junction ligation in one step.● Ultra-fidelity amplification minimizes amplification-preferred steps.Provide your own instruments, kits and consumables1. Magnetic frame: DynaMagTM-2 is recommended.2. DNA purification and recovery kit: It is recommended to use DNA purification and recovery kit by magnetic bead method.3. Library PCR primer kits: transposase method for second-generation sequencing multi-sample primer kits are recommended. 4. Anhydrous ethanol, deionized water (pH between 7.0 and 8.0).5. Reaction tubes: It is recommended to use low adsorption PCR tubes and 1.5 ml centrifuge tubes. Tips: It is recommended to use high quality filter tips to prevent contamination of kits and library samples. Pre-experiment Preparation and Important Notes1. Avoid repeated freezing and thawing of reagents.2. PCR products are easily contaminated due to improper operation, resulting in inaccurate results. It is recommended to isolate the PCR reaction system preparation area from the PCR product purification area, and to use special pipettes to clean the experimental areas at regular intervals.3. Bead purification: the beads should be equilibrated to room temperature before use, all operations on the beads should be carried out at room temperature, 80% ethanol should be dispensed freshly, the beads should be rinsed and dried until the surface is free of liquid reflections and has a frosted appearance, insufficient drying of the beads will cause ethanol residue that will affect the subsequent experiments, and over-drying of the beads will affect the efficiency of DNA recovery.4. The kit is suitable for human genomic DNA library construction, if the DNA sample is a PCR product, it should be ensured that its length>.500 bp, since transposases do not work on DNA ends, it is recommended to extend the PCR product by 50-100 bp at each end of the PCR product to avoid low coverage of the ends for sequencing.Sample PreparationDNA purity requirements: A260/A280 = 1.8-2.0. Sample DNA: dissolve in ultrapure water. DNA Quantification: Too much or too little DNA will affect the quality of the library. It is recommended to use Nano to test the purity of the genomic DNA and then use Qubit to test the concentration of the genome (do not use any absorbance-based assay for template quantification).Schematic diagram of DNA banking processprocedureDNA fragmentation, junction reaction1. Add the following reagents to a 200 µl PCR tube: 2. Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.3. Place the above PCR tubes in the PCR instrument with the hot cap on and program the reaction as follows:DNA should be purified immediately after the fragmentation reaction has been performed and the transposase is still in a high state of activity.to prevent smaller library fragments due to DNA over-fragmentation. Purification of fragmentation productsWe recommend the use of the Century Magnetic Bead Method DNA Purification and Recovery Kit.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. Add 50 µl of magnetic beads equilibrated to room temperature to the fragmentation product, vortex and shake for 5 seconds, then let stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant solution until the solution is clear (approximately 3-5 minutes), carefully aspirate the supernatant and discard, avoiding contact with the beads that have bound the target DNA. Note: Do not discard the beads.4. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the centrifuge tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.5. Repeat step 4.6. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, then add 23 µlddH2O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.7. Remove the tube from the magnetic rack, vortex to completely resuspend the beads, and allow to stand at room temperature for 5 minutes. Centrifuge briefly, place the tube on the magnetic rack until the solution is clear, and transfer 21 µl of supernatant to a new 200 µl PCR tube.PCR amplification Add the following reagents to the 200 µl PCR tube: Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.3. Place the above PCR tubes in the PCR instrument with the hot cap on and program the reaction as follows:Selective recovery of library DNA fragmentsIt is recommended to use CombiVision Magnetic Beads DNA Purification and Recovery Kit for selective recovery of DNA fragments. When different sizes of DNA fragments are required, the amount of magnetic beads to be used is different, please refer to the attached table for the specific amount of magnetic beads to be used (if other brands of magnetic beads are used, you need to find out the optimal amount of magnetic beads to be used on your own).Note: Amplification products can also be fragment length sorted and purified using the Gum Recovery Kit. If there is no special requirement for library length distribution, the amplification products can also be purified without selective recovery of DNA fragments as described on page 6 of the manual.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. Transfer the PCR product to a 1.5 ml centrifuge tube, rehydrate to 100 µl and add several volumes of magnetic beads equilibrated to room temperature, vortex for 5 seconds and let stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant until the solution is clear, and carefully aspirate the supernatant and transfer it to a new 1.5 ml centrifuge tube.Note: Do not discard the top clear.4. Add several volumes of magnetic beads to the supernatant, vortex and shake for 5 seconds, then let stand at room temperature for 5 minutes.5. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant until the solution is clear, carefully aspirate the supernatant and discard it, avoiding contact with the beads that have bound the target DNA.Note: Do not discard the beads.6. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.7. Repeat step 6 once.8. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, add 20 µl of ddH2O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.9. Remove the centrifuge tube from the magnetic rack, vortex and oscillate to completely resuspend the beads, and let stand at room temperature for 5 minutes. Leave brieflycentrifuge, place the tube on a magnetic rack until the solution is clear, and transfer the supernatant solution to a new centrifuge tube. Table: Suggested amount of magnetic beads for different segment selection recoveryLibrary DNA fragment purificationWe recommend the use of the Century Magnetic Bead Method DNA Purification and Recovery Kit.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. 50 µl of magnetic beads equilibrated to room temperature were added to the PCR product, vortexed and shaken for 5 seconds, and then left to stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant solution until the solution is clear (approximately 3-5 minutes), carefully aspirate the supernatant and discard it, avoiding contact with the beads that have bound the target DNA. Note: Do not discard the beads.4. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the centrifuge tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.5. Repeat step 4.6. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, add 25 µl of ddH2O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.7. Remove the tube from the magnetic rack, vortex to completely resuspend the beads, and allow to stand at room temperature for 5 minutes. Centrifuge briefly, place the tube on the magnetic rack until the solution is clear, and transfer the supernatant solution to a new tube.Library quality controlDetermination of library concentrationIn order to obtain high-quality sequencing results, accurate quantification of DNA libraries is required, and the first recommendation is to use Real-timePCR methods are used for absolute quantification of DNA libraries. Additionally, fluorescent dye methods such as the Qubit method or the fluorescent dye picogreen method can be used; do not use quantification methods based on absorbance measurements here. The following approximate formula can be used to convert the molar concentration of the DNA library. Average total length of librariesApproximate conversion formula Library fragment distributionThe prepared DNA libraries can be detected by agarose gel electrophoresis or Agilent 2100 Bioanalyzer.Range of segment length distributions... Read More | Product contentN666081Component50 TStorageN666081ANc-Buffer A50 mL2-8℃N666081BNc-Buffer B3 mL2-8℃N666081CNc-Buffer C25 mL2-8℃N666081DProtease Inhibitor Cocktail750 µL-20℃. Avoid freeze/thaw cycle.ProductsThe Nc-Nucleus/Plasma Protein Extraction Kit is a simple and rapid Product contentN666081Component50 TStorageN666081ANc-Buffer A50 mL2-8℃N666081BNc-Buffer B3 mL2-8℃N666081CNc-Buffer C25 mL2-8℃N666081DProtease Inhibitor Cocktail750 µL-20℃. Avoid freeze/thaw cycle.ProductsThe Nc-Nucleus/Plasma Protein Extraction Kit is a simple and rapid method for extracting nucleus and plasma proteins from mammalian cells and tissues, and the extracted proteins remain biologically active. The kit first cleaves the cell membrane and releases plasma proteins using the plasma protein extraction reagent, and then centrifuges the nucleus to obtain a nucleus precipitate. Finally, the nuclear proteins are extracted by the nuclear protein extraction reagent. The extracted nuclear and plasma proteins are of high purity, effectively avoiding cross-contamination of nuclear and plasma proteins, and can be used for subsequent operations such as Western, Gel Shift, reporter gene detection and enzyme activity determination.Caveat1. If phosphorylated proteins are to be extracted, add a phosphatase inhibitor to the extraction reagent.2. All sample handling should be done on ice.3. The amount of reagents can be adjusted according to the specific experimental situation to ensure that the ratio of each reagent used is Nc-Buffer A:Nc-Buffer B:Nc-Buffer C = 100:5.5:50.4. Higher speeds can be used for centrifugation.ProcedureI Extraction of cytoplasmic and cytosolic proteins from cells1. Please remove the extraction reagents Nc-Buffer A and Nc-Buffer C for pre-cooling before protein extraction.2. Collect the cells and count them. Centrifuge to remove supernatant.3. 1×107 cells were added with 1 ml of Nc-Buffer A (added to Protease Inhibitor Cocktail at a ratio of 1:99 within 2-3 minutes prior to protein pumping), vortexed for 5 seconds to mix well, and incubated on ice for 20 minutes.Note: The characteristics of various cells are different, and the amount of Nc-Buffer A needs to be adjusted according to the characteristics of different cells. If the protein concentration is small, reduce the amount of Nc-Buffer A and subsequent Nc-Buffer B and Nc-Buffer C proportionally.4. Add 55 µl of Nc-Buffer B, vortex for 5 seconds to mix thoroughly, and incubate on ice for 1 minute.5. Centrifuge at 12,000 rpm (~13,400 x g) for 15 minutes at 4°C, collect the supernatant (as clean as possible) into a new centrifuge tube and store at -20°C (this extract is cytoplasmic protein).6. Add 500 µl of Nc-Buffer C (add Protease Inhibitor Cocktail at a ratio of 1:99 before use) to the precipitate obtained in the previous step, vortex for 5 seconds to mix thoroughly, resuspend the precipitate and incubate on ice for 40 minutes, vortexing and mixing at 10-minute intervals for about 15-30 seconds each time.7. Centrifuge at 12,000 rpm for 15 minutes at 4°C, collect the supernatant (as clean as possible) into a new centrifuge tube and store at -20°C (this extract is for cytosolic proteins).II Extraction of cytoplasmic and cytosolic proteins from tissues1. Sampling and preservation of tissues.2. Remove the extraction reagents Nc-Buffer A and Nc-Buffer C for pre-cooling before protein extraction.3. Weigh the tissue and add 1 ml of Nc-Buffer A per 100 mg of tissue (add Protease Inhibitor Cocktail 2-3 minutes before protein extraction at a ratio of 1:99), homogenize well on ice with a homogenizer, and incubate on ice for 20 minutes.Note: The characteristics of various tissues are different, and the amount of Nc-Buffer A needs to be adjusted according to different tissues. If the protein concentration is small, reduce the amount of Nc-Buffer A and subsequent Nc-Buffer B and Nc-Buffer C proportionally.4. Add 55 µl of Nc-Buffer B, vortex for 5 seconds to mix thoroughly, and place on ice for 1 minute of incubation.5. Centrifuge at 12,000 rpm for 15 minutes at 4°C, collect the supernatant (as clean as possible) into a new centrifuge tube and store at -20°C (this extract is cytoplasmic protein).6. Add 500 µl of Nc-Buffer C (add Protease Inhibitor Cocktail at a ratio of 1:99 before use) to the precipitate obtained in the previous step, vortex for 5 seconds to mix thoroughly, resuspend the precipitate and incubate on ice for 40 minutes, vortexing and mixing at 10-minute intervals at, each time for about 15-30 seconds.7. Centrifuge at 12,000 rpm for 15 minutes at 4°C, collect the supernatant (as clean as possible) into a new centrifuge tube and store at -20°C (this extract is cytosolic protein)... Read More | O665690 Component 50T Storage O665690A DNase I 1000 U -20℃.Avoid freeze/thaw cycle. O665690B 10×Reaction Buffer 1000 µL -20℃.Avoid freeze/thaw cycle. O665690C Buffer RLS 40 mL RT O665690D Buffer RW1 40 mL RT O665690E Buffer RW2 (concentrate) 11 mL RT O665690F RNase-Free Water O665690 Component 50T Storage O665690A DNase I 1000 U -20℃.Avoid freeze/thaw cycle. O665690B 10×Reaction Buffer 1000 µL -20℃.Avoid freeze/thaw cycle. O665690C Buffer RLS 40 mL RT O665690D Buffer RW1 40 mL RT O665690E Buffer RW2 (concentrate) 11 mL RT O665690F RNase-Free Water 10 mL RT O665690G Spin Columns FS with Collection Tubes 50 EA RT O665690H Spin Columns RM with Collection Tubes 50 EA RT O665690I RNase-Free Centrifuge Tubes (1.5 mL) 50 EA RTProduct IntroductionThis kit is suitable for extracting RNA from a wide range of plants, even from plants rich in polysaccharides and polyphenols, high quality RNA can be successfully extracted, such as rice leaves, wheat leaves, corn leaves, tobacco leaves, pine needles, ginkgo leaves, poplar leaves, pomegranate leaves, holly leaves, apples, peaches, pears, tomatoes, cherries, apricots, bananas, grapes, loquats, cinnamon rinds, cinnamon pulp, lychee fruit rinds, lychee pulp, soybean, peanut, corn, potato tuber, moonflower petal, pomegranate petal, shiitake mushroom, flat mushroom and other samples. The unique lysate formula can rapidly inactivate the RNA enzyme in the cell, effectively remove the effect of polysaccharide and polyphenol on RNA extraction, without the need for phenol, chloroform and other reagents, while using silicon matrix membrane adsorption of RNA for purification, the total RNA extracted is highly pure, without the contamination of genomes, proteins and other impurities, and can be used for Real Time RT-PCR, RT-PCR, It can be used for Real Time RT-PCR, RT-PCR, Northern Blot, Dot Blot, in vitro translation and other downstream experiments.RNA yieldSelf-contained reagents: β-mercaptoethanol, anhydrous ethanol (freshly opened or for RNA extraction)Pre-experiment Preparation and Important Notes1. To prevent RNase contamination, attention should be paid to the following aspects:1) Use RNase-free plastics and tips.(2) Operators wear disposable masks and gloves, and change gloves diligently during the experiment.2. Avoid repeated freezing and thawing of the extracted samples, otherwise it will affect the rate and quality of RNA extraction.3. If Buffer RLS produces a precipitate, heat to dissolve it and leave at room temperature.4. Please add β-mercaptoethanol to Buffer RLS before use, add 20µl β-mercaptoethanol to 1ml Buffer RLS. Buffer RLS with β-mercaptoethanol can be stored for 1 month at room temperature.5. Anhydrous ethanol should be added according to the instructions on the reagent bottle label before using Buffer RW2 for the first time. Operation steps1. Homogenization: Take 50-100mg of plant tissue and quickly grind it into powder in liquid nitrogen, add 500µl of Buffer RLS (please check whether β-mercaptoethanol is added before use), and immediately mix it by vortexing with vigorous shaking.Note: For materials that are extremely rich in water content, such as watermelon pulp, tomato, pear pulp, etc., more material can be added appropriately, up to 200 mg; for starch-rich samples or mature leaves, the amount of Buffer RLS can be increased appropriately, up to 700 µl.2. Centrifuge at 12,000 rpm (~13,400 x g) for 2 min at 4°C.3. Transfer the supernatant into the filter columns (Spin Columns FS) that have been loaded into the collection tubes, centrifuge at 12,000 rpm at 4°C for 1 minute, carefully aspirate the supernatant in the collection tubes and transfer it to new RNase-Free centrifugation tubes (self-provided), avoiding the tip of the gun from touching the cell debris precipitation in the collection tubes as much as possible.4. Slowly add 0.5 times the volume of the supernatant in anhydrous ethanol, mix well (a precipitate may appear), and transfer the resulting solution together with the precipitate to a Spin Columns RM in a collection tube, or in two batches if you cannot add all of the solution at once. centrifuge the column for 1 minute at 12,000 rpm at 4°C. Dispose of the spent solution and place the column back into the collection tube. Centrifuge at 12,000 rpm for 1 minute at 4°C, discard the spent solution and return the column to the collection tube.5. Add 350 µl of Buffer RW1 to the adsorbent column RM, centrifuge at 12,000 rpm at 4°C for 1 min, discard the waste solution and put the adsorbent column back into the collection tube.6. Preparation of DNase I mixture: Take 52µl of RNase-Free Water, add 8µl of 10×Reaction Buffer and 20µl of DNase I (1U/µl) to it, mix well, and prepare a final volume of 80µl of reaction solution.7. Add 80µl of DNase I mixture directly to the adsorption column and incubate at 20-30°C for 15 minutes.8. Add 350 µl of Buffer RW1 to the adsorbent column RM, centrifuge at 12,000 rpm at 4°C for 1 min, discard the waste solution and put the adsorbent column back into the collection tube.9. Add 500 µl of Buffer RW2 to the adsorbent column RM (check that anhydrous ethanol is added before use), centrifuge at 12,000 rpm for 1 minute at 4°C, discard the waste solution and put the adsorbent column back into the collection tube.10. Repeat step 9.11. Centrifuge at 12,000 rpm for 2 minutes at 4°C.Note: The purpose of this step is to remove residual ethanol from the adsorption column; ethanol residue can interfere with subsequent enzymatic reactions (zymography, PCR, etc.).12. Load the adsorption column RM into new RNase-Free Centrifuge Tubes (1.5 ml), add 30-50 µl of RNase-Free Water dropwise to the middle part of the adsorption membrane overhang, leave it at room temperature for 2 min, and centrifuge at 12,000 rpm at 4°C for 1 min, and store the resulting RNA solution at -70°C to prevent degradation.Note: 1) The volume of RNase-Free Water should not be less than 30 µl, too small volume affects the recovery rate.2) If you want to increase the RNA yield, repeat step 12 with 30-50 µl of fresh RNase-Free Water.3) If the RNA concentration is to be increased, the resulting solution can be reintroduced into the adsorption column and step 12 repeated... Read More | Product content: S665546Component50 TStorageS665546ABuffer QSL45 mLRTS665546BBuffer RIL11 mL2-8℃S665546CBuffer ML10 mLRTS665546DBuffer GW1 (concentrate)13 mLRTS665546EBuffer GW2 (concentrate)26 mLRTS665546FBuffer EBL13 mLRTS665546GRNase A240 µLRTS665546HLysis Tubes Ⅱ50 Product content: S665546Component50 TStorageS665546ABuffer QSL45 mLRTS665546BBuffer RIL11 mL2-8℃S665546CBuffer ML10 mLRTS665546DBuffer GW1 (concentrate)13 mLRTS665546EBuffer GW2 (concentrate)26 mLRTS665546FBuffer EBL13 mLRTS665546GRNase A240 µLRTS665546HLysis Tubes Ⅱ50 EARTS665546ISpin Columns DM With Collection Tubes50 EARTProduct IntroductionThis kit provides a method for extracting total DNA from soil or fecal samples, including the total DNA of cells, bacteria, parasites, and viruses in the samples. It is also suitable for extracting DNA from samples containing high concentrations of PCR reaction inhibitors. This reagent kit adopts a unique buffering system to efficiently bind DNA from the lysis solution to the adsorption column. Inhibitors of PCR and enzyme reactions, as well as residual impurities, can be effectively removed through washing steps. Finally, high-purity DNA can be obtained by washing with low salt buffer or water. The purified DNA can be directly used for downstream experiments such as second-generation sequencing (16S amplicons and metagenomes), library construction, PCR, qPCR, Southern Blot, enzyme digestion molecular markers, etc.Self prepared reagents1. Constant temperature mixer - Product number: CW25932. Anhydrous ethanol, isopropanol3. Vortex oscillator or tissue grinderPreparation and important precautions before the experiment1. Samples should avoid repeated freeze-thaw cycles, otherwise it may result in smaller extracted DNA fragments and a decrease in extraction volume.2.Before the first use, anhydrous ethanol should be added to Buffer GW1 (concentrate) and Buffer GW2 (concentrate) according to the instructions on the reagent bottle label.3. Take out the buffer RIL before use and store it at 2-8 ℃ immediately after use.Operation steps1. Centrifuge the Lysis Tube briefly to allow the beads to settle at the bottom.2. a. Add 0.1-0.3 g of soil or fecal sample to Lysis Tube, and add 740-820 µ L Buffer QSL and 4 µ L RNase A, tighten the tube cover and briefly vortex to mix.b. If fecal samples are stored in non lytic fecal preservation solutions (such as CWY041S and CWY041M), add 200 to Lysis Tube µ L-600 µ L solid-liquid mixture, centrifuge at 13000 rpm for 1 minute, discard the storage solution (if the amount of solid after centrifugation is too small, it can be enriched again, but should not exceed 0.3g). Join 620 µ LBuffer QSL and 4 µ L RNase A, tighten the tube cover and briefly vortex to mix.3. Fix the Lysis Tube in an oscillating grinding device equipped with a 2 mL adapter and process it according to the optimized grinding conditions of your equipment (see appendix).4. Shake the Lysis Tube on a constant temperature mixer at 70 ℃ and 1200 rpm for 10 minutes. Subsequently, centrifuge at 13000 rpm for 2 minutes to precipitate solid particles. Transfer 540 µ Transfer the supernatant to a new 2 mL centrifuge tube.5. Add 180 µ L Buffer RIL, vortex for 5 seconds, centrifuge at 13000 rpm for 2 minutes.Attention: Remove the buffer RIL before use and store it at 2-8 ℃ immediately after use.6. Add 160 to the new centrifuge tube in sequence µ L Buffer ML, 480 µ Supernatant from step 5, 320 µ L isopropanol, vortex for 5 seconds.7. Transfer the solution from the previous step to 650 µ Centrifuge at 12000 rpm (~13400 × g) for 1 minute into the spin columns DM that have been loaded into the collection tube.8. Discard the waste liquid in the collection pipe and place the adsorption column back into the collection pipe. Repeat step 7 until all the solution has been transferred.9. Add 500 to the adsorption column µ L Buffer GW1 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.10. Add 500 to the adsorption column µ L Buffer GW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. 11. Repeat step 10.12.12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes to thoroughly air dry.Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).13. Place the adsorption column in a new centrifuge tube (self provided) and add 50-200 drops of suspended droplets to the middle of the adsorption column µ L Buffer EBL or sterilized water, leave at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 1 minute, collect DNA solution, and store DNA at -20 ℃.Note: 1) Incubating at room temperature for 5 minutes before centrifugation can increase yield.2) Use an additional 50-100 µ Further elution with L buffer or sterilized water can increase yield.3) If you want to increase the final concentration of DNA, you can add the DNA eluent obtained in step 13 back onto the adsorption membrane and repeat step 13, but it may reduce the total yield.4) The elution buffer does not contain chelating agents, please store DNA at -20 ℃.5) The residual trace PCR inhibitors in the genomic DNA template may have adverse effects on the PCR reaction, which can usually be resolved by diluting the DNA by 2-10 times.Appendix: Grind the sample using one of the following methods1. Manually vortex oscillate at maximum speed on the vortex oscillator for 10 minutes.2. On a vortex oscillator equipped with a 1.5-2 mL horizontal centrifuge tube holder, oscillate at maximum speed for 10 minutes (keeping the Lysis Tube horizontal). If the sample size exceeds 12, extend by 5-10 minutes. For example, using Scientific Industries or Mobile's Vortex Genie2 vortex oscillator.3.When using Qiagen's TissueLyser II, grind at 25Hz for 10 minutes.4.When using Qiagen's PowerLyzer 24 Homogenizer, homogenize at 2000 rpm for 30 seconds, pause for 30 seconds, and then homogenize again at 2000 rpm for 30 seconds.5.When using FastPrep-24 from MP Biomedicals, the recommended speed is 6.0 and the time is 40 seconds... Read More |