| 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 | Inquire | DescriptionRefer to the product′s Certificate of Analysis for more information on a suitable instrument technique. Contact Technical Service for further support | Inquire | Product contentU665751Component100 TStorageU665751A2×UltraSYBR One Step Buffer1.4 mL-20℃. Avoid freeze/ Thaw cycle. Protect from light.U665751BUltraSYBR One Step EnzymeMix50 µL-20℃. Avoid freeze/ Thaw cycle. Protect from light.U665751C50×High ROX50 µL-20℃. AvoidProduct contentU665751Component100 TStorageU665751A2×UltraSYBR One Step Buffer1.4 mL-20℃. Avoid freeze/ Thaw cycle. Protect from light.U665751BUltraSYBR One Step EnzymeMix50 µL-20℃. Avoid freeze/ Thaw cycle. Protect from light.U665751C50×High ROX50 µL-20℃. Avoid freeze/ Thaw cycle. Protect from light.U665751DRNase-Free Water1.5 mL-20℃. Avoid freeze/ Thaw cycle. Product Introduction This product is a specialized kit for one-step Real-Time RT-qPCR. The SYBR Green I fluorescent dye contained can bind to all double-stranded DNA, allowing this product to be used for the detection of many different target sequences without the need to synthesize specific labeling probes. Real Time RT-qPCR reaction using this product, reverse transcription and quantitative PCR are carried out in the same reaction system, there is no need to add reagents during the reaction, no need to open the cap of the tube, avoiding contamination while improving the efficiency of the experiment. The new high-efficiency reverse transcriptase RNase H is activity-deficient, which reduces the degradation of RNA in the reverse transcription reaction. The enzyme has high reverse transcription efficiency and can perform a good reverse transcription reaction on a small amount of RNA template. It has high affinity to RNA and can read through RNA templates with high GC content and complex secondary structure. New efficient hot start enzyme, the enzyme activity is closed at room temperature, thus effectively avoiding non-specific amplification caused by non-specific binding of primers and templates or primer dimerization at room temperature, which greatly improves the accuracy of fluorescence quantitative PCR reaction. The included buffer system maximizes the efficacy of both enzymes at the same time and improves efficiency. This product has high sensitivity, high specificity, wide linear range, and more accurate quantification of target genes.ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used with 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 (U665567) Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96 and others. Instruments that require High ROX calibration (U665751) ABI Prism 7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, and others.matters needing attention1. Before using the reagents in this kit, please mix them gently by turning them up and down to avoid foaming as much as possible, and use them after brief centrifugation.2. This product uses RNA as the template for one-step RT-PCR experiment, RNase contamination should be avoided during operation, it is recommended to operate RNA in a special area, use special instruments and consumables, the operator with a mask and disposable gloves and often change the gloves, the experiment-related consumables should be processed with 0.1% DEPC (diethyl ether of pyrocarbonate) aqueous solution at 37℃ for 12 hours and autoclaved for 30 minutes before use. Sterilize for 30 minutes before use.3. UltraSYBR One Step RT-qPCR Buffer contains SYBR Green I fluorescent dye. Avoid bright light when storing this product or preparing PCR reaction solutions.4. Repeated freezing and thawing of each reagent in this kit should be avoided; repeated freezing and thawing may degrade the product performance. This product can be stored for a long time at -20℃, protected from light. If frequent use is required in the short term, it can be stored at 2-8℃.5. This kit must use specific primers, the choice of primers can be selected according to specific experiments, the good or bad primer design directly affects the results of RT-PCR reaction, the design of primers need to consider the GC content, primer length, primer position, the secondary structure of the PCR product and other factors, it is recommended to use a professional primer design software for design.6. This product cannot be used for fluorescent quantitative PCR by the probe method.Usage1. Dissolve RNA template, primers, 2× UltraSYBR One Step Buffer, UltraSYBR One Step EnzymeMix and RNase-Free Water and set aside on ice.2. PCR reaction system:Reagents25 µl Reaction systemFinal concentration2×UltraSYBR One Step Buffer12.5 µl1×Forward Primer，10 µM0.5 µl0.2 µM¹⁾Reverse Primer，10 µM0.5 µl0.2 µM¹⁾UltraSYBR One Step EnzymeMix0.5 µl RNA TemplateX µl10 pg – 100 ng50×Low ROX or High ROX（optional）2）0.5 µl1×RNase-Free Waterup to 25 µlNote: 1) Usually, the primer concentration of 0.2µM can get better results, and the final concentration of 0.1-0.5µM can be used as a reference for setting the range. If the amplification efficiency is not high, the concentration of primer can be increased; when non-specific reaction occurs, the concentration of primer can be decreased, thus optimizing the reaction system.(2) The excitation optical system varies from instrument to instrument, choose to add 50×Low ROX or 50×High ROX according to the instrument using fluorescence quantification.3. Vortex and shake to mix, centrifuge briefly, and collect the solution at the bottom of the tube.4. RT-qPCR reaction conditions (fluorescence quantitative PCR is a two-step method), this program is based on the ABI 7500 fluorescence quantitative PCR instrument as an exampleNote: 1) It is recommended to use two-step PCR reaction program, if you improve the reaction specificity, you can increase the annealing temperature to 60-64 ℃ as a reference for the setting range; if you do not get good experimental results due to the use of primers with lower Tm values, etc., you can try to carry out three-step PCR amplification.(2) For melting curve analysis, please set up the program recommended by the fluorescence quantitative PCR instrument used, and this program is set up with the ABI 7500 fluorescence quantitative PCR instrument as a reference.RT-qPCR reaction conditions (fluorescence quantitative PCR was a three-step method):Note: 1) For three-step PCR amplification, please use the range of 56℃-64℃ as the setting reference for the annealing temperature.(2) For melting curve analysis, please set up the program recommended by the fluorescence quantitative PCR instrument you are using, this program is ABI750 fluorescent quantitative PCR instrument as a reference setting... 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