| 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 content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | Inquire | Products contentN665968Component96 TStorageN665968Adex N501-N508 Primers for Illumina 8×12 µL-20℃. Avoid freeze/thaw cycle.N665968BIndex N701-N712 Primers for Illumina 12×8 µL-20℃. Avoid freeze/thaw cycle. Products IntroductionThis kit is a companion kit for the Products contentN665968Component96 TStorageN665968Adex N501-N508 Primers for Illumina 8×12 µL-20℃. Avoid freeze/thaw cycle.N665968BIndex N701-N712 Primers for Illumina 12×8 µL-20℃. Avoid freeze/thaw cycle. Products IntroductionThis kit is a companion kit for the transposase-based second-generation sequencing Rapid DNA Library Construction Kit, designed for Illumina platform library construction, which contains 8 primers at the N5 end and 12 primers at the N7 end, which can be used to prepare 96 different bipartite Index libraries. All reagents provided in the kit have been subjected to stringent quality control and functional validation to maximize the stability and reproducibility of library construction. The prepared libraries can be sequenced on Illumina platforms such as HiSeq X-10/4000/2500/2000 and MiSeq. Provide your own instruments, reagents and consumables1. Magnetic frame: DynaMagTM-2 is recommended.2. DNA purification and recovery kit: It is recommended to use Kangwei DNA purification and recovery kit by magnetic bead method.3. DNA building kit: It is recommended to use the Kangwei Century transposase method second-generation sequencing rapid DNA building kit.4. Anhydrous ethanol.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 filtration tips to prevent contamination of reagent kits and library samples. Pre-experiment Preparation and Important NotesPlease centrifuge briefly before opening the cap so that the liquid collects at the bottom of the tube to avoid cross-contamination between different primers.For the use of the CombiVision Second Generation Sequencing Multisample Primer Kit, please follow the CombiVision Second Generation Sequencing Rapid DNA Library Kit protocol.Index N501-N508 Primers for IlluminaIndex N701-N712 Primers for Illumina... Read More | S665948 Component 1 mL 5 mL Storage S665948A 2×SYBR qPCR Master Mix 1 mL 5×1 mL -20℃. Avoid freeze/ Thaw cycle. S665948B qPCR Primer Mix 100 µL 5×100 µL -20℃. Avoid freeze/ Thaw cycle. S665948C DNA Standard 1 100 µL 5×100 µL -20℃. Avoid S665948 Component 1 mL 5 mL Storage S665948A 2×SYBR qPCR Master Mix 1 mL 5×1 mL -20℃. Avoid freeze/ Thaw cycle. S665948B qPCR Primer Mix 100 µL 5×100 µL -20℃. Avoid freeze/ Thaw cycle. S665948C DNA Standard 1 100 µL 5×100 µL -20℃. Avoid freeze/ Thaw cycle. S665948D DNA Standard 2 100 µL 5×100 µL -20℃. Avoid freeze/ Thaw cycle. S665948E DNA Standard 3 100 µL 5×100 µL -20℃. Avoid freeze/ Thaw cycle. S665948F DNA Standard 4 100 µL 5×100 µL -20℃. Avoid freeze/ Thaw cycle. S665948G DNA Standard 5 100 µL 5×100 µL -20℃. Avoid freeze/ Thaw cycle. S665948H 50×High ROX 40 µL 200 µL -20℃. Avoid freeze/ Thaw cycle.Product IntroductionThis product is used for real-time fluorescence quantitative PCR (qPCR) using the product after NGS library construction by dye method (SYBR Green I). The kit provides the reaction mixture, DNA primer mixture, and standards required for the qPCR process, and the reagent system is complete, easy and convenient to operate. The kit uses a new chemically modified high-efficiency hot-start polymerase, the activation of the enzyme needs to be incubated at 95 ℃ for 10 min. the product is highly specific, high amplification efficiency, and able to quickly and accurately quantify the concentration of the constructed library. It is suitable for fluorescent quantitative PCR instruments that do not require ROX as a calibration dye, such as Roche LightCycler 480, Roche LightCyler 96, Bio-radiCyleriQ, iQ5, CFX96.ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used in Real Time PCR amplifiers from ABI, Stratagene, and other companies. The excitation optics vary from instrument to instrument, so the concentration of ROX dye must be matched to the corresponding fluorescence quantitative PCR instrument.Instruments that do not require ROX calibration: Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96, etc.Instruments requiring Low ROX calibration: ABI Prism7500/7500 Fast, QuantStudio®3 System, QuantStudio®5 System, QuantStudio®6 Flex System, QuantStudio®7 Flex System, ViiA 7 System, Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, and others.Instruments requiring High ROX calibration: ABI Prism7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, etc.Note: High Rox and Low Rox are formulated as described in Use 2.Scope of applicationThis product is designed for absolute quantification of the concentration of Illumina platform second-generation sequencing libraries. The end of the library contains Illumin P5 and P7 chip binding sequences, the length of which does not exceed 1kb, and the concentration of which is not less than 0.002pM can be used to perform quantitative experiments with this product. The qPCR Primer Mix provided in the kit contains the following two primer sequences:Primer 1:5'-AAT GAT ACG GCG ACC ACC GA-3' Primer 2: 5'-CAA GCA GAA GAC GGC ATA CGA-3'The primer sequence can be used in advance to confirm whether the library can be amplified by that primer pair.UsageAmplification template preparationThe library samples to be detected were diluted with TE (10 mM Tris-Cl, pH 8.0, 1 mM EDTA), and the concentration after dilution was as close as possible to the range of 0.01-20 pM. 4°C on ice was set aside.qPCR reaction system preparationThe desired cryopreservation reagent is pre-melted completely and mixed by inverting several times before preparation, then centrifuged briefly and set aside.The base reaction system for 20 µl was as follows:Reagent20 µl Reaction system2×SYBR qPCR Master Mix10 µlqPCR Primer Mix 10.8 µlTemplate4 µlddH₂O5.2 µlDescription: High Rox model: add 1 µl High Rox per 50 µl of reaction system;Low Rox model: 1 µl High Rox per 500 µl of reaction system.Prepare a sufficient amount of reaction system mixture according to the need, mix well and add to the reaction wells in a volume of 16 µl per well, add the same volume of TE to the blank control, and then add the prepared standards and diluted samples to the corresponding reaction wells in a volume of 4 µl/well. It is recommended to use 20 µl reaction system, if you need to carry out a smaller system reaction, the system components can be reduced in equal proportion.qPCR reaction programThe annealing temperature should be 60-64°C as a reference for the setting range, and the annealing temperature can be increased when a non-specific reaction occurs.If the average length of the library is greater than 700bp, the annealing/extension time should be increased appropriately.data analysisStandard curve productionThe standard curve was plotted using Ct values in the valid range. The standard curve correlation coefficient R2 should not be less than 0.99 and the slope should lie between -3.1 and -3.6. If the standard curve parameters are not reasonable, it is recommended to repeat the experiment.DNA Standard NameDNA Standard ConcentrationDNA Standard 120 pMDNA Standard 22 pMDNA Standard 30.2 pMDNA Standard 40.02 pMDNA Standard 50.002 pMLibrary Concentration CalculationsThe difference in Ct between the three replicate wells of the experiment should be no more than 0.2, otherwise the invalid data should be deleted or the experiment should be repeated. Do not use the Ct outside the valid Ct range of the standard curve to calculate the concentration of the diluted libraries. Please refer to the data processing Excel of this product for the specific library concentration calculation method.matters needing attentionThese instructions should be read in detail before testing. It should be carried out by personnel with specialized experience or qualified by training.Mix gently by turning up and down, avoid foaming as much as possible, and centrifuge for a short time before use.Avoid repeated freezing and thawing of this product; repeated freezing and thawing may degrade product performance.When preparing reaction solutions, use new or non-contaminated tips and centrifuge tubes to prevent contamination as much as possible... Read More |