| Description | Low-Density Lipoprotein (LDL) in plasma is the main carrier of endogenous cholesterol. It is degraded and metabolized by binding to the LDL receptor (LDL-R) on cell membranes and serves as the primary vehicle for transporting cholesterol to peripheral tissues. However, when LDL, especially oxidized Low-Density Lipoprotein (LDL) in plasma is the main carrier of endogenous cholesterol. It is degraded and metabolized by binding to the LDL receptor (LDL-R) on cell membranes and serves as the primary vehicle for transporting cholesterol to peripheral tissues. However, when LDL, especially oxidized LDL (OX-LDL), is present in excess, the cholesterol it carries accumulates in the arterial walls, increasing the risk of atherosclerosis. Atherosclerosis is the pathological basis and risk factor for the majority of cardiovascular and cerebrovascular diseases.Detection Principle: In a cholesterol assay system containing cholesterol esterase (CHER) and cholesterol oxidase (CHOD), specific surfactants are added to selectively solubilize LDL-C for the determination of LDL-cholesterol. Other lipoproteins (HDL, VLDL, chylomicrons) do not react due to inhibition by the surfactants and sugar compounds, remaining in the form of lipoproteins in the reaction mixture. Based on this principle, LDL-cholesterol can be measured directly. Subsequently, esterase catalyzes the hydrolysis of cholesterol esters to generate Free Cholesterol (FC). FC is oxidized by cholesterol oxidase to produce 4-cholestenone and hydrogen peroxide. Hydrogen peroxide then reacts with 4-aminoantipyrine and other components to produce a red quinoneimine compound, which has a characteristic absorption peak at 546 nm. The LDL-C content is determined by measuring the absorbance at 546 nm.Component96TStorageReagent 118 mL2-8℃. Store in the dark.Reagent 26 mL2-8℃. Store in the dark.Standard1EA2-8℃. Store in the dark.Standard (Powder, 1 vial) Preparation:1. Before use, centrifuge at 8000 g, 4°C for 2 minutes to collect the powder at the bottom of the tube.2. Add 0.1 mL of distilled water to dissolve. Use within one week. The prepared concentration is as indicated on the label.User-Prepared Instruments and Reagents:Mortar (Homogenizer), balance, ice box (ice maker), benchtop centrifuge, adjustable micropipettes, water bath (oven, incubator, metal bath), 96-well plate, centrifuge tubes, microplate reader, distilled water (deionized water or ultrapure water are acceptable), ethanol.Experimental ProcedureIt is recommended to first perform a preliminary test using 1-3 samples with expected significant differences (e.g., different types or groups) to familiarize yourself with the procedure and to determine or adjust sample concentrations based on the preliminary results, preventing unnecessary waste of samples or reagents.1. Sample Extraction1.1 Tissue SamplesWeigh approximately 0.1 g of tissue sample and place it in a mortar. Add 1 mL of ethanol and homogenize in an ice bath. Centrifuge at 12,000 rpm, 4°C or room temperature for 10 minutes. Collect the supernatant for assay.Note: If increasing the sample amount, maintain a tissue mass (g) to ethanol volume (mL) ratio between 1:5 and 1:10.1.2 Liquid SamplesAssay clear liquid samples directly. If turbid, centrifuge and use the supernatant for assay.1.3 Bacterial/Cell SamplesCollect bacteria or cells into a centrifuge tube, centrifuge, and discard the supernatant. Add 1 mL of ethanol per approximately 5 million bacteria/cells. Disrupt the bacteria or cells by sonication in an ice bath (power 200W, pulse 3s on, 10s off, repeat 30 times). Centrifuge at 12,000 rpm, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.*Note: If increasing the sample amount, maintain a bacteria/cell count (10⁴) to ethanol volume (mL) ratio between 500:1 and 1000:1.*2. Assay Steps2.1 Preheat the microplate reader for 30 minutes (or wait for the instrument to complete its self-check). Set the wavelength to 546 nm.2.2 Thaw all reagents to room temperature (25°C). Add reagents sequentially to a 96-well plate as follows:Reagent (µL)Test TubeStandard Tube (once)Blank Tube (once)Sample2.5Standard2.5Distilled Water2.5Reagent 1180180180Mix well and incubate at 37°C for 5 minutes. Read the absorbance at 546 nm for each tube (A1 ).Reagent 2606060Mix well and incubate at 37°C for 10 minutes. Read the absorbance at 546 nm for each tube (A2 ). Calculate ΔA = A2 - A1 for each tube.Note:(1) If the A2 value for the Test Tube is greater than 1, dilute the sample with ethanol. The dilution factor (D) must be substituted into the calculation formula.(2) If ΔA for the Test Tube is lower than ΔA for the Blank Tube, consider increasing the sample volume V1 (e.g., increase the sample volume in the Test Tube and the water volume in the Blank Tube to 5 µL or more, keeping Reagents 1 and 2 volumes unchanged; for the Standard Tube, keep at 2.5 µL and add 2.5 µL distilled water to make up volume) or increasing the sample weight W (e.g., to 0.2 g or more). The changed V1 or W must then be substituted into the calculation formula.3. Calculation of Results3.1 Based on Sample MassDerived Formula:LDL-C (µmol/g weight) = (CStandard × V2 ) × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) ÷ (W × V1 ÷ V) × DSimplified Formula:LDL-C (µmol/g weight) = CStandard × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) ÷ W × D3.2 Based on Protein ContentDerived Formula:LDL-C (µmol/mg prot) = (CStandard × V2 ) × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) ÷ (Cpr × V1 ÷ V) × DSimplified Formula:LDL-C (µmol/mg prot) = CStandard × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) ÷ Cpr × D3.3 LDL-C Content in LiquidsDerived Formula:LDL-C (mmol/L) = (CStandard × V2 ) × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) ÷ V1 × DSimplified Formula:LDL-C (mmol/L) = CStandard × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) × D3.4 Based on Cell CountDerived Formula:LDL-C (nmol/10⁴ cells) = (CStandard × V2 ) × 10³ × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) ÷ (500 × V1 ÷ V) × DSimplified Formula:LDL-C (nmol/10⁴ cells) = 2 × CStandard × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) × DParameter Definitions:CStandard : Concentration as indicated on the label (mmol/L or µmol/mL)V1 : Volume of sample added (0.0025 mL)V: Volume of extraction buffer (ethanol) added (1 mL)V2 : Volume of standard added (0.0025 mL)D: Dilution factor (1 if not diluted)500: Number of cells (in units of 10⁴)W: Sample weight (g)Cpr: Protein concentration of the supernatant (mg/mL); Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended.Precautions1. It is recommended to first perform a preliminary test using 1-3 samples with expected significant differences (e.g., different types or groups) to familiarize yourself with the procedure. Based on the preliminary results, determine or adjust sample concentrations to prevent unnecessary waste of samples or reagents.2. This product is for research use only. Not for use in clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation... 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 | Product contentP666142Component200 TStorageP666142ABuffer P160 mLRTP666142BBuffer P260 mLRTP666142CBuffer N380 mLRTP666142DBuffer PB35 mLRTP666142EBuffer PW (concentrate)25 mLRTP666142FBuffer EB30 mLRTP666142GRNase A (10 mg/mL)600 µLRTP666142HSpin Columns DM with Collection Tubes200 EART Product contentP666142Component200 TStorageP666142ABuffer P160 mLRTP666142BBuffer P260 mLRTP666142CBuffer N380 mLRTP666142DBuffer PB35 mLRTP666142EBuffer PW (concentrate)25 mLRTP666142FBuffer EB30 mLRTP666142GRNase A (10 mg/mL)600 µLRTP666142HSpin Columns DM with Collection Tubes200 EART Product IntroductionThis kit is suitable for extracting 1-5 ml of bacterial solution. Based on the lysis of cells by alkaline lysis method, it adopts a unique silica matrix membrane adsorption technology and reagent formulation, and efficiently and exclusively binds plasmid DNA in solution by centrifugal adsorption columns in a high-salt state, and each adsorption column can adsorb a maximum of 30 µg of plasmid DNA, and removes proteins, genomes, RNAs, and other impurities to the greatest extent possible. The plasmid DNA obtained can be directly used for cell transfection, PCR, digestion, sequencing, ligation and other biological experiments.Self-contained reagent: anhydrous ethanol.Pre-experiment Preparation and Important Notes1. All components can be stably stored in dry, room temperature (15-30℃) environment for 1 year, the adsorption column can be stored at 2-8℃ for a longer period of time, and Buffer P1 with RNase A can be stably stored at 2-8℃ for 6 months.2. Before the first use, add all the RNase A solution into Buffer P1, mix well, and store it at 2-8°C. Before use, leave it at room temperature for a period of time, and then use it after recovering to room temperature.3. Anhydrous ethanol should be added to Buffer PW according to the instructions on the label of the reagent bottle before first use.4. If precipitation is found in Buffer P2, Buffer N3, or Buffer PB before use, the clarification can be restored by water bath at 37℃ for a few minutes (please do not shake Buffer P2 violently).5. Be careful not to touch Buffer P2, Buffer N3 and Buffer PB directly, and tighten the lid immediately after use.6. The amount and purity of extracted plasmid is related to the concentration of bacterial culture, strain type, plasmid size, plasmid copy number and other factors.Procedure1. Take 1-5 ml of the overnight culture and add it to a centrifuge tube (self-prepared), centrifuge for 30 seconds at 13,000 rpm (~16,200×g) to collect the bacterial precipitate, and discard the supernatant as much as possible.2. Add 250 µl of Buffer P1 to the centrifuge tube with the bacterial precipitate (please check that RNase A has been added first), mix well using a pipette or vortex shaker, and suspend the bacterial precipitate.Note: If the bacterial mass is not thoroughly mixed, it will affect the lysis effect, resulting in low extraction and purity.3. Add 250µl of Buffer P2 to the centrifuge tube and mix gently up and down 4-6 times, mixing well to lyse the organisms, at which point the solution should become clear and viscous.Note: Mix gently, do not shake vigorously to avoid interrupting the genomic DNA and causing the extracted plasmid to be mixed with genomic DNA fragments. This step should take no more than 5 minutes to avoid damage to the plasmid.4. Add 350 µl of Buffer N3 to the centrifuge tube and immediately mix gently up and down for 8-10 times, mixing well so that a white flocculent precipitate should appear. centrifuge at 13,000 rpm for 5 minutes.Note: Buffer N3 should be mixed immediately after addition to avoid localized precipitation.5. Transfer the supernatant obtained in step 4 to the Spin Columns DM that have been loaded into the collection tube, centrifuge at 13,000 rpm for 30 seconds, pour off the waste liquid from the collection tube, and place the column back into the collection tube.6. Add 150 µl Buffer PB to the adsorption column and centrifuge at 13,000 rpm for 30 seconds.7. Add 400 µl Buffer PW to the adsorption column (please check that anhydrous ethanol has been added first), centrifuge at 13,000 rpm for 1 minute, and pour off the waste liquid in the collection tube.8. Place the adsorbent column in a new centrifuge tube (supplied), add 50-100 µl Buffer EB to the middle of the adsorbent membrane, leave it at room temperature for 2 minutes, centrifuge at 13,000 rpm for 1 minute, and collect the plasmid solution into the centrifuge tube. -The plasmid solution was collected into the centrifuge tube.Note: 1) To increase the recovery efficiency of the plasmid, the resulting solution can be reintroduced into the adsorbent column, left at room temperature for 2 minutes, centrifuged at 13,000 rpm for 1 minute, and the plasmid solution collected into a centrifuge tube.2) For low plasmid copy number or >10 kb, Buffer EB is preheated at 65-70°C in a water bath to increase extraction efficiency... Read More | This reagent kit uses an adsorption column that can specifically bind to viral RNA and a unique buffer system, suitable for isolating viral RNA from cell-free body fluids such as serum, plasma, urine, cerebrospinal fluid, and cell culture supernatants. The viral RNA specifically binds to the siliconThis reagent kit uses an adsorption column that can specifically bind to viral RNA and a unique buffer system, suitable for isolating viral RNA from cell-free body fluids such as serum, plasma, urine, cerebrospinal fluid, and cell culture supernatants. The viral RNA specifically binds to the silicon substrate membrane, and pollutants flow through the membrane. Completely remove impurities such as proteins through two efficient washes, and then wash high-purity viral RNA with RNase free water or RNase Free Water provided by the reagent kit. The virus RNA extracted by this kit can be directly used for experiments such as RT-PCR, Real time RT-PCR, and Western blot analysis. R666005Component50 TStorageR666005ABuffer GL15 mLRTR666005BBuffer RW140 mLRTR666005CBuffer RW2（concentrate）11 mLRTR666005DProteinase K12.5 mgRTR666005EProteinase K Storage Buffer1.25 mLRTR666005FRNase-Free Water10 mLRTR666005GSpin Columns RS with Collection Tubes50 setsRTR666005HRNase-Free Centrifuge Tubes（1.5 mL）50 EART Self prepared reagent: anhydrous ethanol, 0.9% NaCl.Preparation and important precautions before the experiment1. Add 1.25 ml of Protein K Storage Buffer to Protein K to dissolve it and store at -20 ℃. The prepared Protein K should not be left at room temperature for a long time to avoid repeated freeze-thaw cycles, which may affect its activity.2. To prevent RNase pollution, attention should be paid to the following aspects:1) Use RNase free plastic products and gun heads to avoid cross contamination.2) Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use, while plastic containers can be soaked in 0.5 M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.3) Prepare the solution using water without RNase.4) Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.3. Serum or plasma should avoid repeated freeze-thaw cycles that may cause protein denaturation or precipitation, reduce viral titers, and thus affect the yield of extracted viral nucleic acids.4. Before the first use, anhydrous ethanol should be added to Buffer RW2 according to the instructions on the reagent bottle label.5. If buffer GL precipitates, it can be heated at 56 ℃ to dissolve and then placed at room temperature.6. All centrifugation steps should be carried out at room temperature unless otherwise specified, and all operation steps should be carried out quickly.Operation steps1. Take 200 at room temperature µ Add serum or plasma to a 1.5 ml centrifuge tube (self provided). Attention: Less than 200 µ 0.9% NaCl (provided by the customer) can be added to make up for it.2. Add 20 to the solution in the previous step µ Protein K, mix well.3. Add 200 µ L Buffer GL, vortex oscillation for 15 seconds. Note: Do not directly add Protein K to Buffer GL. 4. Incubate at 56 ℃ for 15 minutes, briefly centrifuge, and collect the solution on the tube wall to the bottom of the tube.5. Add 250 µ Anhydrous ethanol, vortex for 15 seconds, incubate at room temperature for 5 minutes, briefly centrifuge, and collect the solution from the tube wall to the bottom of the tube.6. Add all the solution obtained in step 5 to the Spin Columns RS that have been loaded into the collection tube. If it is not possible to add all the solution to the adsorption column at once, please transfer it in two batches, centrifuge at 12000 rpm (~13400 × g) for 1 minute, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.7. Add 500 to the adsorption column µ Centrifuge at 12000 rpm for 1 minute, discard the waste liquid from the collection tube, and place the adsorption column back into the collection tube.8. Add 500 to the adsorption column µ Buffer RW2 (check if anhydrous ethanol is 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.9. Add 500 to the adsorption column µ Centrifuge anhydrous ethanol at 12000 rpm for 1 minute, discard the waste liquid from the collection tube, and place the adsorption column back into the collection tube. 10. Centrifuge at 12000 rpm for 3 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.Attention:1) The purpose of this step is to remove residual ethanol from the adsorption column, which will affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).2) Recommended steps: Place the adsorption column into a new 1.5 ml centrifuge tube (provided), open the tube cover, and incubate in a 56 ℃ oven for 3 minutes to thoroughly dry the membrane of the adsorption column.11. Place the adsorption column in a new RNase free centrifuge tube and add 20-50 to the middle of the adsorption column in the air µ Place RNase Free Water at room temperature for 5 minutes, centrifuge at 12000 rpm for 1 minute, collect RNA solution, and store RNA at -70 ℃ to prevent degradation.Attention:1) The volume of RNase Free Water should not be less than 20 µ l. Small volume affects the recovery rate.2) If you want to increase RNA production, you can use 20-50 µ Repeat step 11 for the new RNase Free Water.3) If you want to increase the RNA concentration, you can add the obtained solution back to the adsorption column and repeat step 11... 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 |