| Description | N665925 Component 1 mL 5 mL Storage N665925A 2×SYBR qPCR Master Mix 1 1 mL 5 mL -20℃. Avoid freeze/ Thaw cycle. N665925B qPCR Primer Mix 1 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665925C DNA Standard I 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665925 Component 1 mL 5 mL Storage N665925A 2×SYBR qPCR Master Mix 1 1 mL 5 mL -20℃. Avoid freeze/ Thaw cycle. N665925B qPCR Primer Mix 1 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665925C DNA Standard I 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665925D DNA Standard II 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665925E DNA Standard III 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665925F DNA Standard IV 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665925G DNA Standard V 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665925H 50×High ROX 40 µL 200 µL -20℃. Avoid freeze/ Thaw cycle.Product IntroductionThis product is a real-time fluorescence quantitative PCR of the products after NGS library construction using a dye method (SYBR Green I).(qPCR). The kit provides the reaction mixes, DNA primer mixtures, standards and sample dilutions required for the qPCR process, making the reagent system complete and easy to use. The fluorescent dye SYBR Green I contained in the reaction mixture can bind to all double-stranded DNA; the GoldStar Taq DNA Polymerase used is a chemically modified new high-efficiency hot-start polymerase, and the activation of the enzyme needs to be incubated at 95℃ for 10 minutes. the product has high specificity, high amplification efficiency, and is able to quickly and accurately quantify the concentration of the constructed libraries. The product is highly specific and efficient in amplification, and can quickly and accurately quantify the concentration of the constructed library.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 Ion torrent platform second generation sequencing libraries. The end of the library contains Ion torrent P5 and P7 microarray binding sequences, the length of which does not exceed 1kb, and the concentration is not less than 0.005pM 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'-CCA TCT CAT CCC TGC GTG TC - 3' Primer 2: 5'-CCT CTC TAT GGG CAG TCG GTG AT-3'The primer sequence can be used in advance to confirm whether the library can be amplified by that primer pair.Usage1. Amplification 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 0.05-50 pM. 4°C on ice was set aside.2. 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 MasterMix10 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.3. qPCR reaction program1) Please use 60-64℃ as a reference for setting range of annealing temperature, and increase the annealing temperature when non-specific reaction occurs.2) If the average length of the library is greater than 700bp, the annealing/extension time should be increased appropriately.data analysis1. Standard 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 I50 pMDNA Standard II5 pMDNA Standard III0.5 pMDNA Standard IV0.05 pMDNA Standard V0.005 pM2. Library concentration calculationThe 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 attention1. Before testing, these instructions should be read in detail. It should be operated by personnel with professional experience or qualified by training.2. For use, please mix gently by turning up and down, avoid foaming as much as possible, and use it after centrifugation for a short period of time.3. Avoid repeated freezing and thawing of the product, repeated freezing and thawing may degrade the performance of the product.4. When preparing the reaction solution, please 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 | DescriptionCAR10 is a kit that contains a selection of 10 carbohydrates/sugars: Arabinose, Fructose, Galactose, Glucose, α-Lactose, Maltose, Mannose, Ribose, Sucrose and Xylose, which may be used for general research, as reagents or as reference compounds in analytical procedures | Product content:D665967Component200 TStorageD665967ABuffer PB120 mLRTD665967BBuffer PS60 mLRTD665967CBuffer PW (concentrate)25 mLRTD665967DBuffer EB30 mLRTD665967ESpin Columns DM with Collection Tubes200 EART Product Introduction: This reagent kit adopts a new silicon-based membrane technology and Product content:D665967Component200 TStorageD665967ABuffer PB120 mLRTD665967BBuffer PS60 mLRTD665967CBuffer PW (concentrate)25 mLRTD665967DBuffer EB30 mLRTD665967ESpin Columns DM with Collection Tubes200 EART Product Introduction: This reagent kit adopts a new silicon-based membrane technology and reagent formula. Through a rapid and simple three-step process of binding, washing, and elution, 100 bp-10 kb DNA fragments can be purified and recovered from PCR products or enzyme reaction solutions (enzyme cutting, linking, probe labeling, etc.). Each adsorption column can adsorb up to 10 kb of DNA fragments µ G DNA, while minimizing impurities such as primers, oligonucleotides, enzymes, etc. The purified and recovered DNA has high purity and concentration, good integrity, and high recovery rate, and can be directly used for molecular biology experiments such as sequencing, linking and transformation, labeling, and in vitro transcription.Self prepared reagent: anhydrous ethanol.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. When the solution is stored at low temperature, it should be left at room temperature for a period of time before use, and then restored to room temperature before use.2. This reagent kit can selectively recover all DNA fragments from the solution. If you need to selectively recover specific fragments while removing other fragments of different sizes, please choose our company's gel recovery reagent kit.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 the Buffer PB. If there is any crystallization or precipitation, you can take a water bath at 37 ℃ for a few minutes to restore clarity.5. The recovery efficiency is related to the initial amount of DNA and the elution volume. The smaller the initial amount, the smaller the elution volume, and the lower the recovery rate.6. All centrifugation steps can be performed at room temperature.Operation steps:1. Estimate the volume of DNA reaction solution, add 5 times the volume of Buffer PB, and mix thoroughly (without removing paraffin or mineral oil).Note: 1) If the DNA reaction system is 50 µ l (excluding paraffin oil volume), add 250 µ l Buffer PB.2) After adding Buffer PB, check the pH value of the solution. If the pH value is greater than 7.5, add 10-30 to it µ 3 M sodium acetate (pH 5.0) was used to adjust the pH value to 5-7.2. Column balance: Add 200 to the spin columns DM that have been loaded into the collection tube µ Centrifuge at 13000 rpm (~16200 × g) for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.3. Add the solution obtained in step 1 to the adsorption column that has been loaded into the collection tube, let it stand at room temperature for 1 minute, centrifuge at 13000 rpm for 30-60 seconds, discard the waste liquid in the collection tube, and place the adsorption column in the collection tube.Attention: The volume of the adsorption column is 750 µ l. If the sample volume is greater than 750 µ l, it can be added in batches.4. Add 500 µ l of Buffer PW to the adsorption column (please check if anhydrous ethanol has been added before use), centrifuge at 13000 rpm for 30-60 seconds, discard the waste liquid in the collection tube, and place the adsorption column in the recovery tube.Note: If purified DNA is used for salt sensitivity experiments (such as flat end ligation experiments or direct sequencing), it is recommended to add Buffer PW and let it stand for 2-5 minutes before centrifugation.5.13000 rpm for 1 minute 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.). To ensure that downstream experiments are not affected by residual ethanol, it is recommended to open the cover of the adsorption column and place it at room temperature for a few minutes to thoroughly dry the residual ethanol in the adsorbent material at the bottom.6. Place the adsorption column into a new centrifuge tube (provided by oneself), add 30-50 µ l Buffer EB to the middle position of the adsorption membrane by hanging droplets, and let it stand at room temperature for 1 minute. Centrifuge at 13000 rpm for 1 minute and collect DNA solution- Store DNA at 20 ℃.Attention:1) 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 (the pH value of water can be adjusted to this range using NaOH).2) To improve the recovery of DNA, the solution obtained by centrifugation can be added back to the adsorption column, left at room temperature for 2 minutes, and centrifuged at 13000 rpm for 1 minute.3) The elution volume should not be less than 30 µ l. A small volume will affect the recovery efficiency... Read More | N665917 Component 1 mL 5 mL Storage N665917A 2×SYBR qPCR MasterMix 1 mL 5×1 mL -20℃. Avoid freeze/ Thaw cycle. N665917B qPCR Primer Mix 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917C DNA Standard A 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917 Component 1 mL 5 mL Storage N665917A 2×SYBR qPCR MasterMix 1 mL 5×1 mL -20℃. Avoid freeze/ Thaw cycle. N665917B qPCR Primer Mix 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917C DNA Standard A 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917D DNA Standard B 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917E DNA Standard C 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917F DNA Standard D 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917G DNA Standard E 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917H 50×High ROX 40 µL 200 µL -20℃. Avoid freeze/ Thaw cycle.Product IntroductionThis is a dye-based (SYBR Green I) qPCR NGS library quantification kit for cfDNA, which provides the reaction mixture, DNA primer mixture, standards, and sample dilutions required for the qPCR process, making it a complete reagent system that is easy and convenient to use. The fluorescent dye SYBR Green I contained in the reaction mixture binds to all double-stranded DNA. 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, the length of the standard in the kit (about 270bp) is comparable to the average length of the cfDNA NGS libraries (250-300bp), which is able to quickly and accurately quantitate the concentration of the constructed cfDNA libraries. quantification.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, ViiA7 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 Method of Use 2.Applicable scopeThis product is designed for the absolute quantification of the concentration of Illumina platform second generation sequencing libraries. The end of the library contains Illumina P5 and P7 microarray binding sequences, the length of which does not exceed 1kb, and the concentration is not less than 0.02pM can be used for quantitative experiments. 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 0.01-60 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 MasterMix10 µlqPCR Primer Mix0.8 µlTemplate4 µlddH₂O5.2 µlDescription: High Rox model: 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.3.qPCR reaction programIf the average length of the library is greater than 700bp, the annealing/extension time should be increased appropriately.Refer to the specific instrument setup program for dissolution curves.data analysisStandard curve productionThe standard curve was plotted according to the data processing Excel sheet. The correlation coefficient R2 of the standard curve should be not less than 0.99, and the slope should be located between -3.1 and -3.6 when the Ct value is the longitudinal coordinate. If the parameters of the standard curve are unreasonable, it is recommended to repeat the experiment.DNA Standard NameDNA Standard ConcentrationDNA Standard A60 pMDNA Standard B6 pMDNA Standard C0.6 pMDNA Standard D0.06 pMDNA Standard E0.006 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 |