| Description | Phosphoenolpyruvate Carboxykinase (PEPCK, EC 4.1.1.32) is widely present in animals, plants, microorganisms, and cells. It catalyzes the conversion of oxaloacetate to phosphoenolpyruvate and is a key regulatory enzyme in the gluconeogenesis pathway.Assay PrinciplePEPCK catalyzes the conversion of Phosphoenolpyruvate Carboxykinase (PEPCK, EC 4.1.1.32) is widely present in animals, plants, microorganisms, and cells. It catalyzes the conversion of oxaloacetate to phosphoenolpyruvate and is a key regulatory enzyme in the gluconeogenesis pathway.Assay PrinciplePEPCK catalyzes the conversion of Oxaloacetate to Phosphoenolpyruvate and CO₂. Pyruvate Kinase and Lactate Dehydrogenase subsequently catalyze the sequential oxidation of NADH to NAD⁺. The rate of decrease in NADH absorbance at 340 nm is measured, which reflects PEPCK activity.Component100TStorageExtraction Buffer100 mL2-8℃Reagent 118 mL2-8℃Reagent 216.5 µL2-8℃Reagent 31EA-20℃Reagent 41EA-20℃Required Materials and Equipment (Not Provided)Spectrophotometer / Microplate reader, benchtop centrifuge, adjustable pipettes, micro quartz cuvette / 96-well plate, mortar and pestle, ice, and distilled water.Sample Preparation:1.Bacteria or Cultured Cells:Collect cells by centrifugation and discard the supernatant.Add Extraction Buffer at a ratio of 1 ml per 5-10 million cells (e.g., 1 ml for 5 million cells).Sonicate on ice (20% power or 200W, pulse 3s on/10s off, repeat 30 times).Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.2.Tissues:Homogenize tissue on ice in Extraction Buffer at a ratio of 1:5-10 (w/v) (e.g., 0.1 g tissue in 1 ml buffer).Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.3.Serum (or Plasma) Samples:Assay directly.Assay Procedure:1.Preheat the spectrophotometer or microplate reader for at least 30 minutes. Set the wavelength to 340 nm. Zero the instrument with distilled water.2.Preparation of Working Solution: Just before use, transfer and dissolve Reagent 2 and Reagent 3 into Reagent 1. Mix well. Aliquot and store any unused portions at -20°C. Avoid repeated freeze-thaw cycles.3.Preparation of Reagent 4: Just before use, dissolve the contents of the vial in 1 ml of distilled water. Mix well. Aliquot and store any unused portions at -20°C. Avoid repeated freeze-thaw cycles.4.Pre-warm the Working Solution and dissolved Reagent 4 at 37°C (for mammalian samples) or 25°C (for other species) for 5 minutes.5.In a micro quartz cuvette or a well of a 96-well plate, add:10 µl sample10 µl dissolved Reagent 4180 µl pre-warmed Working SolutionMix immediately and record the initial absorbance (A₁) at 340 nm. Record the absorbance again (A₂) after exactly 1 minute. Calculate ΔA = A₁ - A₂.Note: For this kit, if ΔA is greater than 0.1, dilute the sample with Extraction Buffer by an appropriate factor (account for this dilution factor 'n' in the calculations) so that ΔA is less than 0.1 to improve detection sensitivity.PEPCK Activity Calculation:1. Calculation for Micro Quartz Cuvette (d = 1.0 cm)General Parameters for Cuvette:Vₜₒₜₐₗ (Total reaction volume) = 0.0002 L (200 µL)ε (NADH molar extinction coefficient) = 6220 L/mol/cmd (Cuvette light path) = 1.0 cmVₛₐₘₚₗₑ (Sample volume in reaction) = 0.01 mL (10 µL)T (Reaction time) = 1 minVₛₐₘₚₗₑₜₒₜₐₗ (Total extract volume) = 1 mL (for tissues/cells)Cpr (Sample protein concentration, mg/mL)W (Sample mass, g)500 (Cell/Bacteria count in millions for example calculation: 5 million)a. For Serum (Plasma):Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per ml of serum.Calculation:PEPCK Activity (nmol/min/ml) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ Vₛₐₘₚₗₑ ÷ TSimplified Formula: PEPCK (nmol/min/ml) = 3215 × ΔAb. For Tissues, Bacteria, or Cells:Based on Sample Protein Concentration:Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per mg of protein.Calculation:PEPCK Activity (nmol/min/mg prot) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ × Cpr) ÷ TSimplified Formula: PEPCK (nmol/min/mg prot) = 3215 × ΔA ÷ CprBased on Sample Fresh Weight:Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per gram of fresh tissue.Calculation:PEPCK Activity (nmol/min/g fresh weight) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (W × Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ TSimplified Formula: PEPCK (nmol/min/g fresh weight) = 3215 × ΔA ÷ WBased on Bacterial or Cell Density:Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per 10⁴ cells.Calculation (example for 5 million cells in 1 ml extract):PEPCK Activity (nmol/min/10⁴ cell) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (500 × Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ TSimplified Formula: PEPCK (nmol/min/10⁴ cell) = 6.43 × ΔA2. Calculation for 96-Well Plate (d = 0.5 cm)General Parameters for 96-Well Plate:(All parameters remain the same except for the light path 'd')d (96-well plate light path) = 0.5 cma. For Serum (Plasma):Simplified Formula: PEPCK (nmol/min/ml) = 6430 × ΔAb. For Tissues, Bacteria, or Cells:Based on Sample Protein Concentration:Simplified Formula: PEPCK (nmol/min/mg prot) = 6430 × ΔA ÷ CprBased on Sample Fresh Weight:Simplified Formula: PEPCK (nmol/min/g fresh weight) = 6430 × ΔA ÷ WBased on Bacterial or Cell Density:Simplified Formula: PEPCK (nmol/min/10⁴ cell) = 12.86 × ΔAPrecautionsBefore formal assay, it is essential to perform a pilot test with 2-3 samples expected to have significant differences in activity... Read More | Product introduction：This kit uses uniqcell lysis and heme / protein precipitation technology, combined with DNA preparation membrane to selectively adsorb DNA to achieve the purpose of purifying genomic DNA.Scope of application：Nucleic acid extraction and purification | EndoFree Plasmid Midi Kit Cat No. Component Size（50T） Storage E665631A Buffer P1 30 mL RT E665631B Buffer P2 30 mL RT E665631C Buffer E3 30 mL RT E665631D Buffer PS 15 mL RT E665631E Buffer PW (concentrate) 10 mL RT E665631F Endo-free Buffer EB 10 mL RTEndoFree Plasmid Midi Kit Cat No. Component Size（50T） Storage E665631A Buffer P1 30 mL RT E665631B Buffer P2 30 mL RT E665631C Buffer E3 30 mL RT E665631D Buffer PS 15 mL RT E665631E Buffer PW (concentrate) 10 mL RT E665631F Endo-free Buffer EB 10 mL RT E665631G RNase A (10 mg/mL) 600 µL RT E665631H Buffer ER 8 mL RT E665631I CWBlue 300 µL RT E665631J Spin Columns DL with Collection Tubes 50 EA RT E665631K Endo-Remover FM with Collection 50 EA RTProduct Introduction:Endotoxins are a common pollutant in plasmid extraction. Due to the high sensitivity of eukaryotic cells to endotoxins, the presence of endotoxins in plasmids can greatly reduce the transfection efficiency of eukaryotic cells. This reagent kit provides a simple, fast, and efficient new method for extracting endotoxin free plasmids. The extracted plasmids can remove endotoxins to the maximum extent possible and effectively remove contamination of genomic DNA, RNA, proteins, and other substances. This reagent kit is suitable for extracting 5-15mL of bacterial solution. On the basis of alkaline lysis of cells, it efficiently and specifically binds plasmid DNA through a new silicon-based membrane. Each adsorption column can adsorb up to 100 µ The plasmid DNA of g is effectively removed using a special buffer system and endotoxin removal filter column, effectively removing impurities such as endotoxins and proteins. The plasmid obtained from this kit has high purity and stable quality, making it particularly suitable for cell transfection. It can also be used for downstream experiments such as DNA sequencing, PCR, PCR based mutations, in vitro transcription, transformed bacteria, and endonuclease digestion.Self prepared reagents: anhydrous ethanol, isopropanol.Preparation and important precautions before the experiment:1. All components can be stably stored in a dry, room temperature (15-30 ℃) environment for 1 year, and can be stored at 2-8 ℃ for longer periods of time. Buffer P1 with RNase A added can be stably stored at 2-8 ℃ for 6 months.2. Before the first use, add all RNase A solution to Buffer P1, mix well, and store at 2-8 ℃. Before use, let it sit at room temperature for a period of time. After returning to room temperature, use.3.Before the first use, anhydrous ethanol should be added to the Buffer PW according to the instructions on the reagent bottle label.4. Before use, please check if there is any crystallization or precipitation in Buffer P2 and Buffer E3. If there is any crystallization or precipitation, you can take a water bath at 37 ℃ for a few minutes to restore clarity.5. Be careful not to come into direct contact with Buffer P2 and Buffer E3, and immediately cover them tightly after use.6.The amount and purity of plasmid extraction are related to factors such as bacterial culture concentration, strain type, plasmid size, and plasmid copy number.Operation steps:1. Take 5-15 mL of overnight cultured bacterial solution and add it to a centrifuge tube (self provided). Centrifuge at 13000 rpm (~16200 × g) for 1 minute to collect bacteria, and try to discard all the supernatant as much as possible.2. Add 500 to the centrifuge tube containing bacterial sediment µ L Buffer P1 (please check if RNase A has been added first), mix thoroughly with a pipette or vortex oscillator, and suspend bacterial precipitation. Attention: If the bacterial blocks are not thoroughly mixed, it will affect the cracking effect, resulting in low extraction amount and purity.3. Add 500 to the centrifuge tube µ L Buffer P2, gently invert and mix 8-10 times, allowing the bacterial cells to fully lyse. Leave at room temperature for 3-5 minutes. At this point, the solution should become clear and viscous. Attention: Mix gently and do not shake vigorously to avoid interrupting genomic DNA and mixing genomic DNA fragments in the extracted plasmid. If the solution does not become clear, it indicates that the bacterial count may be too high and the lysis may not be complete. The bacterial count should be reduced.4. Add 500 to the centrifuge tube µ L Buffer E3, immediately invert and mix 8-10 times until white flocculent precipitates appear. Let it stand at room temperature for 5 minutes. Centrifuge at 13000 rpm for 5 minutes, extract the supernatant, and add it to the filter column (Endo Remove FM) (already loaded into the collection tube). Centrifuge at 13000 rpm for 1 minute to filter, then transfer the filtrate from the collection tube to the centrifuge tube (self provided). Attention: 1) After adding Buffer E3, it should be immediately mixed to avoid local precipitation. 2) The maximum volume of the adsorption column is 750 µ L. So please filter the supernatant twice and mix it in the same self provided centrifuge tube.5. Add 450 to the filtrate µ Mix L isopropanol upside down.6. Column balance: Add 200 to the spin columns DL that have been loaded into the collection tube µ L Buffer PS, centrifuge at 13000 rpm for 2 minutes, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.7. Transfer the mixed solution of filtrate and isopropanol from step 5 to an equilibrium adsorption column (already loaded into a collection tube). 8.13000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. Attention: The maximum volume of the adsorption column is 750 µ L. So the solution obtained in step 5 is divided multiple times and passed through the column. 9. Add 750 to the adsorption column µ L Buffer PW (please check if anhydrous ethanol has been added first), centrifuge at 13000 rpm for 1 minute, and discard the waste liquid in the collection tube.10. Place the adsorption column back into the recovery manifold and centrifuge at 13000 rpm for 1 minute.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.).11. Place the adsorption column in a new centrifuge tube (self provided)... Read More | Inquire | 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 |