| Description | Glycogen is a macromolecular polysaccharide composed of glucose and serves as one of the primary storage forms of sugar. It is mainly stored in the liver and muscles as reserve energy, referred to as liver glycogen and muscle glycogen, respectively. Liver glycogen regulates blood glucose Glycogen is a macromolecular polysaccharide composed of glucose and serves as one of the primary storage forms of sugar. It is mainly stored in the liver and muscles as reserve energy, referred to as liver glycogen and muscle glycogen, respectively. Liver glycogen regulates blood glucose concentration; when blood sugar rises, glycogen can be synthesized in the liver, and when blood sugar decreases, liver glycogen is broken down into glucose to supplement blood sugar. Therefore, liver glycogen is crucial for maintaining the relative balance of blood glucose. Muscle glycogen is the storage form of sugar in muscles. During strenuous exercise that consumes large amounts of blood sugar, muscle glycogen cannot be directly broken down into blood sugar but must first decompose to produce lactic acid, which circulates to the liver via the bloodstream and is converted into liver glycogen and glucose through gluconeogenesis. Detection Principle: Glycogen is extracted using a strong alkaline extraction buffer. Under strong acidic conditions, it forms a blue compound with the anthrone chromogen, which has a characteristic absorption peak at 620 nm. Within a certain concentration range, the glycogen content is linearly related to the absorbance at 620 nm. The glycogen content in the sample can be calculated based on the standard curve. Detection Range: 0.003125 - 0.25 mg/mL Sensitivity: 0.003125 mg/mL Applicable Samples: Animal tissues, bacteria, cellsG1501748Component96TStorageG1501748AExtraction Buffer120 mL2-8℃G1501748BChromogen1EA2-8℃. Store in the dark.G1501748CStandard1 mL2-8℃Note: It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.User-Provided Instruments and Consumables1.Microplate reader or visible spectrophotometer (capable of measuring absorbance at 620 nm)2.Low-temperature centrifuge, Water bath3.96-well plate or micro glass cuvettes, Adjustable pipettes and tips, EP tubes4.Deionized water, Concentrated sulfuric acidExperimental Procedure1. Reagent PreparationReagent NameReagent PreparationPrecautionsExtraction BufferReady-to-use; equilibrate to room temperature before use.Store at 4°C. Corrosive; please take protective measures during handling.ChromogenFirst, dissolve the powder in 7.2 mL of deionized water. Then slowly add 28.8 mL of concentrated sulfuric acid. Mix thoroughly after complete dissolution.Store at 4°C protected from light; valid for one week. Toxic; please take protective measures during handling.StandardStore at 4°C.2. Standard Curve SetupDilute the 1 mg/mL standard with deionized water to prepare standard solutions of 0.25, 0.1, 0.05, 0.025, 0.0125, 0.00625, and 0.003125 mg/mL as shown in the table below.No.Standard VolumeDeionized Water Volume (µL)Concentration (mg/mL)Std.1100µL of 1mg/mL3000.25Std.2160µL of Std.12400.1Std.3200µL of Std.22000.05Std.4200µL of Std.32000.025Std.5200µL of Std.42000.0125Std.6200µL of Std.52000.00625Std.7200µL of Std.62000.003125Note: A standard curve must be prepared for each experiment. Diluted standard solutions are unstable and must be used within 4 hours.3. Sample PreparationNote: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month.3.1 TissuesWeigh 0.1 g of tissue and place it in a 10 mL test tube. Add 0.75 mL of Extraction Buffer. Boil in a water bath for 20 minutes (stopper the tube tightly to prevent water evaporation). Shake the tube every 5 minutes to mix thoroughly. After the tissue is completely dissolved, remove the tube and let it cool. Dilute to 5 mL with deionized water, mix well. Centrifuge at 8,000 g, 25°C for 10 minutes. Collect the supernatant for detection.3.2 Cells or BacteriaCollect 5 million bacteria or cells into an EP tube. Centrifuge and discard the supernatant. Add 0.75 mL of Extraction Buffer and disrupt the bacteria or cells by ultrasonication (power 200 W, ultrasonicate for 3 s, interval 10 s, repeat 30 times). Transfer to a 10 mL test tube. Boil in a water bath for 20 minutes (stopper the tube tightly to prevent water evaporation). Shake the tube every 5 minutes to mix thoroughly. Remove the tube and let it cool. Dilute to 5 mL with deionized water, mix well. Centrifuge at 8,000 g, 25°C for 10 minutes. Collect the supernatant for detection.Note: For protein concentration determination, Aladdin BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) are recommended.4. Assay Steps4.1 Instrument Preparation: Preheat the microplate reader or visible spectrophotometer for at least 30 minutes. Set the wavelength to 620 nm. For visible spectrophotometers, zero the instrument with deionized water.4.2 Sample Assay: Add reagents sequentially to EP tubes as follows:ReagentBlank Tube (µL)Standard Tube (µL)Test Tube (µL)Sample0060Standard0600Deionized Water6000Chromogen2402402404.3 Mix well. Incubate in a 95°C water bath for 10 minutes (cap tightly to prevent evaporation). Cool. Transfer 200 µL to a 96-well plate or micro glass cuvette. Measure the absorbance at 620 nm, recorded as A blank, A standard, and A test. Calculate ΔA test = A test - A blank and ΔA standard = A standard - A blank. Note: It is recommended to perform preliminary experiments with 2-3 samples expected to have significant differences before formal testing. If ΔA test is less than 0.001, appropriately increase the sample amount. If ΔA test is greater than 1.5, dilute the sample further with deionized water (multiply the result by the dilution factor) or reduce the amount of sample used for extraction. 5. Result Calculation Note: We provide both derived and simplified calculation formulas, which are equivalent. The simplified formulas in bold are recommended as the final calculation formulas. 5.1 Standard Curve Plotting Plot the standard curve with standard concentration as the y-axis and ΔA standard as the x-axis (using concentration as the y-axis facilitates calculation). Substitute ΔA test into x to calculate y (mg/mL). 5.2 Sample Glycogen Content Calculation (1) Based on sample mass: Glycogen (mg/g) = 1.11 × (y × V sample ) ÷ (W × V sample ÷ V total ) × n = 5.55 × y ÷ W × n (2) Based on sample protein concentration: Glycogen (mg/mg prot) = 1.11 × (y × V sample ) ÷ (V sample × Cpr) × n = 1.11 × y ÷ Cpr × n (3) Based on bacterial or cell count: Glycogen (mg/10⁴) = 1.11 × (y × V sample ) ÷ (Bacterial or Cell Count × V sample ÷ V total ) × n = 5.55 × y ÷ Bacterial or Cell Count × n Parameter Description: 1.11: Constant for converting glucose content measured by this method to glycogen content (i.e., 100 µg glucose color developed with anthrone reagent is equivalent to that of 111 µg glycogen). V sample : Volume of test sample added to the reaction system, 0.06 mL. W: Sample mass, g. V total : Total volume of the sample extract, 5 mL. n: Dilution factor. Cpr: Sample protein concentration, mg/mL. Bacterial or Cell Count: In units of 10⁴ (ten thousands)6. Result PresentationTypical Standard Curve: y = 0.1746x + 0.0027, R² = 0.9961(The following data and curve are for reference only; users must establish their own standard curve based on their experiment.)Precautions1. It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.2. This product is for scientific research use only and is not intended for clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation... Read More | H665581 Component 100 T Storage H665581A gDNA Eraser 50 µL -20℃. Avoid freeze/thaw cycle. H665581B 10×gDNA Eraser Buffer 120 µL -20℃. Avoid freeze/thaw cycle. H665581C HiFiScript, 200 U/µL 100 µL -20℃. Avoid freeze/thaw cycle. H665581D 5×ScriptRT H665581 Component 100 T Storage H665581A gDNA Eraser 50 µL -20℃. Avoid freeze/thaw cycle. H665581B 10×gDNA Eraser Buffer 120 µL -20℃. Avoid freeze/thaw cycle. H665581C HiFiScript, 200 U/µL 100 µL -20℃. Avoid freeze/thaw cycle. H665581D 5×ScriptRT Buffer 500 µL -20℃. Avoid freeze/thaw cycle. H665581E Primer Mix 120 µL -20℃. Avoid freeze/thaw cycle. H665581F RNase-Free Water 2×1 mL -20℃. Avoid freeze/thaw cycle.Product IntroductionThis product is a kit for removing genomic DNA for reverse transcription. The kit removes genomic DNA in 2 minutes at 42°C. Since the reverse transcription reagent contains a component that inhibits gDNA Eraser, cDNA can be synthesized directly by reverse transcription of gDNA Eraser-treated samples.The kit is equipped with a new high-efficiency reverse transcription enzyme, HiFiScript, with novel mutation sites that dramatically increase the transcriptional activity of the enzyme, resulting in higher efficiency and yield of cDNA first-strand synthesis. The first strand of cDNA can be synthesized with higher efficiency and yield, and the first strand of cDNA can be synthesized from pg total RNA or mRNA. If the reverse transcription product cDNA is used for downstream fluorescence quantitative detection, the reverse transcription reaction can be completed at 42℃ in 15 minutes. This kit is suitable for the synthesis of first-strand cDNA and subsequent RT-PCR, RT-qPCR, and the construction of full-length cDNA libraries.Product Features1. Rapid genome removal: contains gDNA Eraser for genomic DNA removal, which removes genomic DNA in just 2 minutes.2. Rapid reverse transcription: 15 minutes to obtain fluorescent quantitative PCR template cDNA first strand synthesis.3. High sensitivity: cDNA first strand can be synthesized using pg-level total RNA or mRNA templates.4. Highly efficient reverse transcription: Novel mutation sites dramatically increase enzyme activity, resulting in higher yields of cDNA.matters needing attention1. During operation, RNase contamination should be avoided to prevent RNA degradation or cross-contamination in the experiment. It is recommended that operators wear masks and disposable gloves and change the gloves frequently, and use specialized instruments and consumables.2. The reverse transcription system is prepared and operated on ice to prevent degradation of RNA. Store the kit enzymes at -20ºC as soon as possible after use and try to avoid repeated freezing and thawing.3. The reaction system can be scaled up to a maximum of 1 µg of total RNA in 10 µl of reaction system.4. Primer Mix is prepared by Oligo(dT) and Random primer, and Oligo-dT Primer or Gene Specific Primer can also be used according to the experimental needs.5. If the amount of starting RNA is less than 50ng, it is recommended to add RNAase inhibitor (RNasin).6. For RNA templates with complex secondary structures, it is recommended to incubate the template RNA at 65°C for 5 minutes immediately on ice prior to the manipulation step and centrifuge briefly before proceeding to the next step.UsageThaw template RNA on ice; place kit components on ice immediately after thawing at room temperature. Each solution was mixed by vortexing and shaking before use and briefly centrifuged.I. Genomic DNA removal reactions1. Prepare the reaction system according to the following table on ice in a total volume of 10 µl. To ensure the accuracy of the reaction solution preparation, prepare the premixed system in the amount of reaction number + 2 before dispensing it into each reaction tube and finally adding the RNA sample.Note: 1) If the amount of total RNA is greater than 1µg, scale up the reaction system proportionally. If the amount of starting RNA is less than 50ng, it is recommended to add RNAase inhibitor (RNasin).2. Mix by vortex shaking and centrifuge briefly so that the solution on the walls of the tube collects at the bottom.3. Incubate at 42°C for 2 minutes (this can be extended to 30 minutes for room temperature reactions).4．At the end of the reaction, centrifuge briefly and place on ice to cool.II. Reverse transcription reaction1. Prepare the reaction system on ice according to the following table. In order to ensure the accuracy of the reaction solution configuration, first prepare a premixed solution in the amount of number + 2, and then dispense 10 µl into each reaction tube, take 10 µl of the prepared premixed solution and add it to the reaction tube of step 1 where the de-etching of the genome has been completed.Note: 1) Oligo-dT Primer or Gene Specific Primer can be used according to the needs of the experiment, it is recommended to use 50 pmol of Oligo-dT Primer or 2 pmol of Gene Specific Primer for 20 µl reaction system.2. Mix well and centrifuge briefly so that the solution on the walls of the tube collects at the bottom.3. cDNA synthesis reaction conditions:1) If fluorescent quantitative PCR assay is performed downstream, incubate at 42°C for 15 minutes and 85°C for 5 minutes.2) If downstream for normal PCR assay, incubate at 42°C for 30-50 minutes and 85°C for 5 minutes. Note: For templates with complex secondary structure or high GC content, the reverse transcription temperature can be increased to 50°C to enhance reverse transcription efficiency.4. At the end of the reaction, centrifuge briefly and place on ice before proceeding with subsequent PCR or fluorescence quantitative PCR, or place at -20°C if prolonged storage is required.Note: When performing Real-time PCR reactions, the amount of reverse transcription product added should not exceed 1/10 of the total volume of the PCR reaction... Read More | Products contentProducts IntroductionThis product uses the principle that the difference between the concentration of salt ions inside and outside the cell can cause the cell membrane to burst to lyses the cell and releases the genomic DNA, without the need of extracting and purifying the genomic Products contentProducts IntroductionThis product uses the principle that the difference between the concentration of salt ions inside and outside the cell can cause the cell membrane to burst to lyses the cell and releases the genomic DNA, without the need of extracting and purifying the genomic DNA.This product is suitable for a variety of sources of samples, and can be used as a template for PCR and qPCR experiments after sample processing, and can achieve the effect of the purified DNA used as a template for PCR and qPCR experiments. Usage1. Depending on the type of sample, prepare the appropriate sample size according to the table below.2. Add the sample to a 1.5-mi centrifuge tube and add the recommended volume of Solution A as shown in the table below. Vortex for 20 s and allow to stand at room temperature for 3-5 min or incubate in a metal bath at 95°C for 3-5 min as recommended in the table below.3. After the sample has been sufficiently lysed (samples incubated in a metal bath at 95°C should be brought to room temperature), add the recommended volume of Solution B as shown in the table below and vortex for 30s.4. Store processed samples at 4°C if the next test is to be performed within 2 hours, or at -20°C if the next test cannot be performed immediately.take note of1) Depending on the requirements of the experimental conditions, the amount of samples can be expanded or reduced, and the amount of Solution A and Solution B can be increased in equal proportions.2) For blood and cell samples, the temperature of room temperature lysis is required to be around 25C. If the ambient temperature does not reach 25°, the lysis time can be extended appropriately, or the vortex shaking time can be extended to ensure that the samples are fully lysed. If there is no relevant professional instrument, the centrifuge tube can be shaken vigorously to ensure adequate lysis.3) After the tissue sample is made into tissue homogenate by adding 10 times the volume of saline, it can be processed in the same way as blood samples.4) Strictly prohibit the use of expired products, please do not mix different reagents.5) laboratory supplies should be regularly cleaned and 10% of the 84 disinfectant solution or ultraviolet lamp for anti-pollution treatment, special areas dedicated to prohibit cross use, so as to avoid contamination, the end of the test, the bench should be cleaned immediately... Read More | Product content： Component S665549 50 preps Buffer SW 60 ml Buffer SL 60 ml Buffer GL 50 ml Buffer GW1（concentrate） 2X13 ml Buffer GW2（concentrate） 15 ml Buffer GE 15 ml Spin Columns DM 50 with Collection Tubes 50Product IntroductionThis kit is suitable for Product content： Component S665549 50 preps Buffer SW 60 ml Buffer SL 60 ml Buffer GL 50 ml Buffer GW1（concentrate） 2X13 ml Buffer GW2（concentrate） 15 ml Buffer GE 15 ml Spin Columns DM 50 with Collection Tubes 50Product IntroductionThis kit is suitable for extracting total DNA from fecal samples, including the total DNA of cells, bacteria, parasites, and viruses in the samples, as well as samples containing high concentrations of PCR reaction inhibitors. This product can process up to 300 mg of fecal samples and purify to obtain mainly 20-30 kb DNA fragments. The purification process does not require toxic solvents such as phenol or chloroform, and does not require ethanol precipitation. High purity DNA can be obtained within one hour. This reagent kit adopts a unique buffering system to efficiently bind DNA from the lysis solution to the adsorption column. At the same time, protein impurities and other organic compounds that inhibit downstream reactions in feces can flow through the membrane. Inhibitors of PCR and enzyme reactions, as well as residual impurities, can be effectively removed through two washing steps. Finally, high-purity DNA can be obtained by washing with low salt buffer or water. The purified DNA can be directly used for downstream experiments such as enzyme digestion, PCR, Real Time PCR, library construction, Southern Blot, and molecular labeling.Preparation and important precautions before the experiment1. Samples should avoid repeated freeze-thaw cycles, otherwise it may result in smaller extracted DNA fragments and a decrease in extraction volume.2.Before the first use, anhydrous ethanol should be added to Buffer GW1 and GW2 according to the instructions on the reagent bottle label.3. Before use, please check whether there is crystallization or precipitation in Buffer SL and Buffer GL. If there is crystallization or precipitation, please dissolve Buffer SL and Buffer GL again in a 56 ℃ water bath.4. If downstream experiments are sensitive to RNA contamination, 4 can be added after adding Buffer SL µ RNase A of DNase Free (100 mg/ml) is not provided in this kit. If needed, it can be ordered separately from our company, item number: S665549Operation steps1. Take a fecal sample of 100-300 mg and place it in a centrifuge tube (provided by oneself).2. Add 1 ml of Buffer SW and vortex for 3-5 minutes to evenly disperse the sample in the solution. Centrifuge at 12000 rpm (~13400 × g) for 1 minute and discard the supernatant.3. Add 1 ml of Buffer SL and vortex for 3-5 minutes to evenly disperse the sample in the solution. Take a water bath at 65 ℃ for 20 minutes and vortex for 15 seconds every 5 minutes. Note: To remove RNA, add 4 after completing the above steps µ RNase A solution (product number: CW0601S) with a concentration of 100 mg/ml, shake well and let stand at room temperature for 5-10 minutes.4.Centrifuge at 2000 rpm for 3 minutes and transfer the supernatant to a new centrifuge tube (provided by oneself).5. Add an equal volume of Buffer GL to the supernatant, invert and mix 15-25 times, and leave on ice for 5 minutes. Centrifuge at 12000 rpm for 5 minutes. Attention: At this time, the liquid may be in a transparent or turbid state, which does not affect the experiment. 6. Add the supernatant obtained in step 5 to the spin columns DM that have been loaded into the collection tube. If the solution cannot be added at once, it can be transferred multiple times. Centrifuge at 12000 rpm (~13400 × g) for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.7. Add 500 to the adsorption column µ Buffer GW1 (check if anhydrous ethanol has been 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. 8. Repeat step 7.9. Add 500 to the adsorption column µ Buffer GW2 (check if anhydrous ethanol has been 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.10.12000 rpm for 2 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. 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) and add 50-100 drops of suspended droplets to the middle of the adsorption column µ L Buffer GE or sterilized water, leave at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 1 minute, collect DNA solution, and store DNA at -20 ℃.Note: 1) If downstream experiments are sensitive to pH or EDTA, they can be washed off with sterilized water. The pH value of the eluent has a significant impact on the elution efficiency. If water is used as the eluent, its pH value should be ensured to be between 7.0-8.5 (NaOH can be used to adjust the pH value of the water to this range). When the pH value is below 7.0, the elution efficiency will be reduced2) Incubating at room temperature for 5 minutes before centrifugation can increase yield.3) Use an additional 50-100 µ Further washing with buffer GE or sterilized water can increase yield.4) If you want to increase the final concentration of DNA, you can add the DNA eluent obtained in step 11 back onto the adsorption membrane and repeat step 11; It is possible to increase the final concentration of DNA, but it may reduce the total yield. If the amount of DNA is less than 1 µ g. Recommended 50 µ Wash with buffer GE or sterilized water.5) DNA stored in water can be affected by acidic hydrolysis. If long-term storage is required, it is recommended to elute with Buffer GE and store at -20 ℃.6) The residual trace PCR inhibitors in the genomic DNA template may have adverse effects on the PCR reaction, which can be solved by diluting DNA by 2-10 times... 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 |