| Description | 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 | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | Lipid peroxidation is the degradation of lipids that occurs as a result of oxidative damage and is a useful marker for oxidative stress. Polyunsaturated lipids are susceptible to an oxidative attack, typically by reactive oxygen species, resulting in a well-defined chain reaction with the productionLipid peroxidation is the degradation of lipids that occurs as a result of oxidative damage and is a useful marker for oxidative stress. Polyunsaturated lipids are susceptible to an oxidative attack, typically by reactive oxygen species, resulting in a well-defined chain reaction with the production of end products such as malondialdehyde (MDA). Lipid peroxidation may contribute to the pathology of many diseases including atherosclerosis, diabetes, and Alzheimer′s.Lipid peroxidation (MDA) assay kit has been used to determine the levels of malondialdehyde (MDA).Suitability: Suitable for the measurement of malondialdehyde (MDA) in a variety of samples including tissue, cells and plasmaPrinciple: In this kit, lipid peroxidation is determined by the reaction of MDA with thiobarbituric acid (TBA) to form a colorimetric (532 nm)/fluorometric (λex= 532/λem= 553 nm) product, proportional to the MDA present... Read More | Product introduction：Griess reagent can be used for spectrophotometric detection of nitrite. The reagent contains two chemicals, sulfonic acid and n- (1-naphthyl) ethylenediamine. Under acidic conditions, sulfamic acid is converted into diazonium salt by nitrite, which can form a highly Product introduction：Griess reagent can be used for spectrophotometric detection of nitrite. The reagent contains two chemicals, sulfonic acid and n- (1-naphthyl) ethylenediamine. Under acidic conditions, sulfamic acid is converted into diazonium salt by nitrite, which can form a highly colored azo dye with n- (1-naphthyl) ethylenediamine. This dye can be detected at 548 nm: because no is extremely unstable, it is oxidized to form nitrite and nitrate. Griess indirectly reflects the content of no by detecting the content of nitrite.Matters needing attention：1. before using Griess reagent, return it to room temperature and check the solution for precipitation. If Griess reagent I contains sediment when taken out, it can be placed in a 37 ℃ water bath until the sediment dissolves. 2. this product is potentially harmful. Avoid prolonged or repeated exposure. Avoid entering eyes, skin or clothing. Please wear lab clothes and disposable gloves for operation.Scope of application：No detectionComponent：Instruction：1.Griess Reagent I and II were taken out to restore the room temperature.2.Standard dilution : The standard NaNO2 ( 1-100 µM ) was diluted with the solution used for the sample to be tested. The standard was diluted to 1 µM, 10 µM, 20 µM, 40 µM, 80 µM and 100 µM, and 100 µL standard was added to each well. If the sample concentration is too low, the range of the standard curve can be appropriately reduced ( 1 µM, 2 µM, 3 µM, 4 µM, 6 µM, 8 µM, 10 µM ).3.Sample detection :( 1 ) According to the total volume of 200 µL / hole, 100 µL / hole sample was added to the 96-well plate ; if the sample is the supernatant of the culture medium, it can be sampled directly, and if there is sediment, the supernatant should be taken after centrifugation. If the sample is a cell or tissue, it can be quickly lysed by freeze-thaw, and then centrifuged to obtain the supernatant. The volume of less than 100 µL can be diluted with diH2O or 0.9 % NaCl ( corresponding standards also need to be diluted with diH2O or 0.9 % NaCl ).( 2 ) According to 50 µL / hole, Griess Reagent I was added to each hole.( 3 ) According to 50 µL / hole, Griess Reagent II was added to each hole.( 4 ) The absorbance was measured at 540 nm. If there is no 540 nm filter, 520-560 nm filter can also be. If there is no microplate reader or a suitable filter, the concentration of nitric oxide in the sample can also be determined by visual colorimetry. A more precise concentration gradient is required for the standard when visual colorimetric... 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 |