| Description | Fructokinase (FK, EC 2.7.1.4) regulates the interconversion between sucrose and starch and is involved in the regulation of plant metabolism and growth development. Fructokinase (FK) phosphorylates fructose to generate fructose-6-phosphate. This product is subsequently acted upon by a series of Fructokinase (FK, EC 2.7.1.4) regulates the interconversion between sucrose and starch and is involved in the regulation of plant metabolism and growth development. Fructokinase (FK) phosphorylates fructose to generate fructose-6-phosphate. This product is subsequently acted upon by a series of composite enzymes, reducing NADP+ to NADPH. The enzyme activity of fructokinase is determined by measuring the rate of increase in NADPH absorbance at 340 nm.Component100TStorageExtraction Buffer120 mL2-8℃Reagent 120 mL2-8℃Reagent 21EA-20℃Reagent 31EA-20℃Reagent 41EA2-8℃Reagent 51EA2-8℃Reagent PreparationReagent 2 (Powder, 1 vial):Before use, centrifuge at 8000 g, 4°C for 2 min to collect the powder at the bottom (tap manually if needed).Add 1.1 mL of distilled water to dissolve.The storage period is the same as the kit's expiry date.Reagent 3 (Liquid, 1 vial):Before use, centrifuge at 8000 g, 4°C for 2 min to collect the liquid at the bottom (tap manually if needed).Add 1.05 mL of distilled water to dissolve.The storage period is the same as the kit's expiry date.Reagent 4 (Powder, 1 vial):Before opening, ensure the powder is at the bottom of the vial (tap manually if needed).Add 17 mL of Reagent 1 to dissolve.The storage period is the same as the kit's expiry date.Reagent 5 (Liquid, 1 vial):Before use, centrifuge at 8000 g, 4°C for 2 min to collect the liquid at the bottom (tap manually if needed).Add 1.1 mL of distilled water to dissolve.The storage period is the same as the kit's expiry date.User-Prepared Instruments & MaterialsMortar (homogenizer), ice bucket (ice maker), benchtop centrifuge, adjustable pipettes, water bath (oven, incubator, metal bath), 96-well plate, centrifuge tubes, microplate reader, distilled water (deionized water or ultrapure water is acceptable).Sample Extraction1. Tissue Samples: Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and homogenize on ice. Centrifuge at 12000 rpm, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.Note: If increasing the sample amount, use a ratio of 1:5 to 1:10 (tissue weight (g) : Extraction Buffer volume (mL)) for extraction.2. Bacterial/Cell Samples: Collect bacteria or cells into a centrifuge tube by centrifugation and discard the supernatant. Take 5 million bacteria or cells, add 1 mL of Extraction Buffer, and disrupt using ultrasound on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 10 s, repeat 30 times). Centrifuge at 12000 rpm, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.*Note: If increasing the sample amount, use a ratio of 1:1000~5000 (bacteria/cell count (x10⁴) : Extraction Buffer volume (mL)) for extraction.*3. Liquid Samples: Detect directly. If the sample is turbid, centrifuge and use the supernatant for detection.Assay Procedure1. Preheat the microplate reader for at least 30 minutes. Set the wavelength to 340 nm.2. Thaw all reagents to room temperature (25°C).3. In a well of the 96-well plate, add sequentially:Reagent (µL)Test WellSample20Reagent 210Reagent 310Reagent 4150Mix well and incubate at 37°C for 5 minutes. 4. Add:Reagent (µL)Test WellReagent 5105. Mix well. Immediately read the absorbance at 340 nm (A1) and then read again after incubating at 37°C for 20 minutes (A2). Calculate ΔA = A2 - A1.Notes:1. If ΔA is close to zero, the reaction time can be appropriately extended to 30 minutes or longer before reading A2; or the sample volume V1 can be increased appropriately (with a corresponding decrease in Reagent 4 volume). The modified reaction time (T) and sample volume (V1) must be substituted into the calculation formula.2. If the initial absorbance A1 is too high (e.g., >2, as in deeply pigmented plant leaves), appropriately reduce the sample volume V1. The modified V1 must be substituted into the calculation formula. Alternatively, add a small amount of activated carbon to the sample, mix, let stand for 5 min, then centrifuge at 12000 rpm, 4°C for 10 min, and use the supernatant for detection.3. If the increasing trend is unstable, read the absorbance every 10 seconds and select a linear increasing period for calculation. The corresponding ΔA value should be substituted into the calculation formula.FK Activity Calculation1. Based on Sample Protein Concentration:Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADPH per minute per mg of protein.Formula:FK (nmol/min/mg prot) = [ΔA ÷ (ε × d) × V2 × 10⁹] ÷ (Cpr × V1) ÷ T = 160.77 × ΔA ÷ Cpr2. Based on Sample Fresh Weight:Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADPH per minute per gram of fresh tissue.Formula:FK (nmol/min/g fresh weight) = [ΔA ÷ (ε × d) × V2 × 10⁹] ÷ (W × V1 ÷ V) ÷ T = 160.77 × ΔA ÷ W3. Based on Bacterial/Cell Density:Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADPH per minute per 10⁴ bacteria/cells.Formula:FK (nmol/min/10⁴ cell) = [ΔA ÷ (ε × d) × V2 × 10⁹] ÷ (500 × V1 ÷ V) ÷ T = 0.322 × ΔA4. Based on Liquid Volume:Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADPH per minute per mL of liquid.Formula:FK (nmol/min/mL) = [ΔA ÷ (ε × d) × V2 × 10⁹] ÷ V1 ÷ T = 160.77 × ΔAParameter Description:ε: NADPH molar extinction coefficient, 6.22 × 10³ L/mol/cmd: Light path of the 96-well plate, 0.5 cmV: Volume of Extraction Buffer added, 1 mLV1: Volume of sample supernatant added, 0.02 mLV2: Total reaction volume, 0.2 mL = 2.0 × 10⁻⁴ LT: Reaction time, 20 minW: Sample mass, g500: Cell number, in units of 10⁴Cpr: Protein concentration of the supernatant, mg/mL; Aladdin BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) are recommended.Precautions It is recommended to first select 1-3 samples with significant differences (e.g., different types or groups) for preliminary experiments to familiarize yourself with the procedure. Determine or adjust the sample concentration based on the preliminary results to prevent unnecessary waste of samples or reagents... Read More | The bacterial viability / toxicity detection kit contains two fluorescent dyes. Nucgreen is a green nucleic acid dye that can stain live and dead bacteria; Ethd III is a red nucleic acid dye that only stains dead bacteria with damaged cell membranes. When nucgreen and ethd III are properly mixed, The bacterial viability / toxicity detection kit contains two fluorescent dyes. Nucgreen is a green nucleic acid dye that can stain live and dead bacteria; Ethd III is a red nucleic acid dye that only stains dead bacteria with damaged cell membranes. When nucgreen and ethd III are properly mixed, the bacteria with intact cell membrane appear green, while the bacteria with damaged cell membrane can appear green and red under different channels, respectively. A common criterion for bacterial viability is the ability to propagate in a suitable nutrient medium, known as a growth assay. This kit is generally in good agreement with the growth assay results in liquid or solid medium. However, under certain conditions, membrane damaged bacteria may recover and propagate in nutrient medium, and such bacteria will be identified as dead bacteria in this assay. On the contrary, some bacteria with intact membranes may not be able to propagate in nutrient medium, but will be recognized as viable bacteria in this assay. Therefore, if there is a large difference between the test results of this kit and the bacterial growth assay, the above possibilities should be considered. Component： Product parameters： NucGreen: Ex/Em = 503/530 nm (结合 DNA)；EthD-III: Ex/Em = 530/620 nm (结合 DNA)。Usage:1 Preparation of control samples for live and dead bacteria (optional)1. Cultivate 4 mL of bacteria in liquid medium until late logarithmic phase.2. Prepare two 1 mL bacterial solutions in an EP tube and centrifuge for 10-15 minutes under 5000-10000 g conditions.3. Remove the supernatant and add 0.3 mL of 0.85% NaCl resuspended bacteria to one of the EP tubes, and 1 mL of 0.85% NaCl resuspended bacteria to the other tube.4. Add 0.7 mL of isopropanol to a tube containing 0.3 mL of 0.85% NaCl, and mix thoroughly (with a final concentration of 70% isopropanol) to prepare a dead bacterial sample.5. Incubate the two samples at room temperature for 1 hour and mix every 15 minutes.6. Centrifuge the two samples at 5000-10000 g for 10-15 minutes.7. Remove the supernatant, add 1 mL of 0.85% NaCl to resuspend the bacteria in both samples, and centrifuge again as in step 6.8. Use a spectrophotometer to measure the absorbance values (OD670) of two bacterial suspensions at 670 nm.9. Adjust the density of the two bacterial suspensions (live and dead) to 108 bacteria/mL (OD670 ≈ 0.3), and then dilute with 0.85% NaCl at 1:100 to achieve a final density of 106 bacteria/mL.10. Mix two bacterial suspensions as shown in the table below to obtain the required live cell ratio: dead cell ratio.Table 1 Mix live and dead bacterial suspensions by a certain volume to achieve the required ratio of live and dead cellsLive cells: Dead cellsVolume of viable bacterial suspension（mL）Volume of dead bacterial suspension（mL）0:10001.010:900.10.920:800.20.830:700.30.750:500.50.5100:01.00II Staining methods for fluorescence microscopy observation1. Mix 1 volume of component A, NucGreen, and 2 volumes of component B, EthD-III, in a microcentrifuge tube. After thorough mixing, add 8 volumes of 0.85% NaCl solution to obtain a 100 x dye solution.2. Every 100 µ L bacterial suspension, add 1 µ 100 x dye solution of L.3. Mix thoroughly and incubate at room temperature in the dark for 15 minutes.4. Take 5 µ The bacterial suspension after L staining was dropped onto a glass slide with an 18 mm square cover glass.5. Observe under a fluorescence microscope. The fluorescence of live and dead bacteria can be observed simultaneously under any standard FITC long-acting filter. Alternatively, live (green fluorescent) and dead (red fluorescent) bacteria can be observed using FITC and Cy3 (or Texas Red) channels, respectively.Attention: (1) Before staining bacteria, attention must be paid to removing residues of growth media. Nucleic acid and other media components can bind to NucGreen and EthD-III dyes in some way, resulting in unacceptable staining changes. A simple washing step is usually sufficient to remove interfering media components from bacterial suspension. It is not recommended to use phosphate buffer solutions as they can reduce staining efficiency. (2) Before starting the formal experiment, the dye concentration should be adjusted to distinguish between NucGreen labeling live bacteria and EthD-III labeling dead bacteria. The optimal concentration may vary depending on the bacterial strain. It is generally best to use the lowest dye concentration that can provide sufficient signal. The above conditions have been optimized for staining live/dead cells of Escherichia coli.III Before starting the staining method experiment of flow cytometry, please read the precautions under the fluorescence microscope staining steps.According to Table 1, add 11 different proportions of live and dead bacteria to the EP tube. Each of the 11 samples has a volume of 1 mL.2. Add 12 µ The A component of L, NucGreen, and 24 µ The B component EthD-III of L was mixed in a microcentrifuge tube. Add 3 to each of the 11 samples µ Mix the mixed dyes of L thoroughly by blowing them up and down several times. (Note: Additional control bacterial samples need to be prepared for separate NucGreen and EthD-III staining)3. Incubate at room temperature in the dark for 15 minutes.4. Analyze each sample using a flow cytometer, detect NucGreen positive cells using FITC channels, and detect EthD-III positive cells using PI or PE channels.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. if the orifice plate is used for detection, a small amount of bacterial liquid can be left for imaging after standing for 10 min, which can effectively reduce the background. 3. in order to be closer to the real results, it is recommended to keep the brightness of red fluorescence consistent with that of green fluorescence in merge pictures. 4. fluorescent dyes have quenching problems. Please try to avoid light during experimental operation to slow down fluorescence quenching. 5. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Staining of dead and live bacteria... Read More | Inquire | 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 | Inquire |