| Description | In the experimental process of molecular biology, Fluo ™ Green ® The dsDNA quantification kit is a product used for fluorescence detection and quantification of double stranded DNAThe method is very sensitive. Commonly used in molecular biology techniques: construction of cDNA libraries; In the experimental process of molecular biology, Fluo ™ Green ® The dsDNA quantification kit is a product used for fluorescence detection and quantification of double stranded DNAThe method is very sensitive. Commonly used in molecular biology techniques: construction of cDNA libraries; Purification and application of DNA fragments for subcloning, such as DNA quantification, product amplification, and further detection of primers. Vaccines are a commonly used control method in modern disease prevention. Nowadays, many vaccines are cell culture vaccines, such as recombinant hepatitis B vaccine, rabies vaccine and most other vaccines are produced by cell culture. Among them, the purification of vaccines is a key issue, and we need to remove host cell DNA and host proteins as much as possible. If the DNA and protein of the host cell are injected into the human body together with the vaccine, unpredictable consequences will occur.The conventional method for detecting DNA content is to measure its absorbance at 260nm (A260). The main disadvantage of this method is that nucleotides, single stranded nucleic acids, and proteins have a significant impact on the signal, and are also subject to interference from pollutants during the nucleic acid preparation process, making it difficult to distinguish between DNA and RNA. Additionally, this method is insensitive (5 µ g/mL dsDNA solution A260=0.1). Fluo ™ The Green quantitative detection method is simple and convenient, and has been selected by multiple biological product factories, becoming the standard for residual DNA detection in biological products.At present, this method has been included in the 2010 edition of the Chinese PharmacopoeiaPrinciple:Fluo ™ Green emits fluorescence only after binding to double stranded DNA, and does not emit fluorescence without DNA; The fluorescence emitted is directly proportional to the concentration of DNA. In the 2010 Chinese Pharmacopoeia, it was proposed that, Fluo ™ The detection limit of Green's quantitative DNA method is about 0.3ng/ml, and the linearity is good (R2>0.99) when the DNA content is in the range of 1.25-80ng/mLAdvantage:1) This method can determine double stranded DNA from any expressed host sample.2) It is possible to directly quantify PCR amplification products without purifying DNA from the reaction mixture.3) Far exceeding the sensitivity of traditional UV A260 detection methods and Hoechst33258.4) Higher concentrations of salt, urea, ethanol, chloroform, detergents, proteins, or agarose have no effect on the measurement.5) The effect of measuring dsDNA in the presence of equimolar concentrations of ssDNA and RNA is minimal.Required equipment• Micro fluorescence meter; Portable fluorescence analyzer - Shanghai Huguo Scientific Instrument Co., Ltd. HG-9 model; 1cm quartz colorimetric dish• Fluo ™ Green dsDNA quantitative detection kit, 1mL of concentrated reagent solution is sufficient for 200 measurements of 2mL volume.1×TE（10mM Tris 1mM EDTA）pH8.0； 250ug/mL calf thymus DNAExperimental planPreparation of reagentsFluo ™ The Green dsDNA quantification reagent is stored in anhydrous DMSO (dimethyl sulfoxide) in the form of 1mL concentrated solution. On the day of the experiment, prepare2X Fluo ™ The operating solution of Green's reagent was diluted with 1xTE at a ratio of 1:200 in concentrated solution (10mM Tris HCl,1mM EDTA, pH 7.5). If you want to prepare enough operating solution to determine 20 samples, you can add 100 µ L Fluo to 20mL1x TE ™ Green dsDNA quantification reagent. Due to the easy adsorption of reagents onto glass surfaces, they need to be prepared in plastic containers. Fluo ™ Green reagent is easily degraded by light, so the prepared solution should be wrapped in foil or stored in a dark place away from light.It is best to use the solution within a few hours of preparation to ensure optimal results.Experimental method:1). Preparation of standard working fluid:1mg of calf thymidine DNA dry powder (Tris, NaCl, and other concentrations have become standard systems) is added to 1mL of double distilled water to prepare a 1mg/mL standard working solution;2). Configuration of dye working fluid:6 uL Fluo ™ Add 1mL of TE to Green (note: use 1 × TE to mix Fluo) ™ Dilute Green 200 times, use and prepare immediately, avoid light.3). Dilution of standard working fluid:(1) Mother liquor dilution: Take 10ul (1mg ⁄ mL) of standard working solution and add it to 990ul TE solution to dilute the concentration to 10ug ⁄ mL. Take 10ul (10ug ⁄ mL) of standard working solution and add it to 990ul TE solution to dilute the concentration to 100ng ⁄ mL;(2) Dilute by multiple ratio: Take 800ul (100ng ⁄ mL) of standard working solution and add it to 200ul of TE solution to achieve a concentration of 80ng ⁄ mL (pharmacopoeia regulation: fluorescent)The light staining method shows good linearity in the range of 1.25-80 ng/mL for DNA content, with a detection limit of 0.3 ng/mL. Take 500ul (80ng ⁄ mL) of standard working solution and add it to 500ul TE solution, diluting the concentration to 40ng ⁄ mL; Dilute sequentially by multiple ratios to prepare 20ng/ml 10ng/ml 5.0ng/ml 2.5ng/ml1.25ng/ml and 0.625ng/ml standard solution;4). Preparation of standard curve: Take 100ul of each gradient standard solution and dye working solution diluted by multiple ratios, mix well, and leave them at room temperature in the dark for 5 minutes. Use FB-15 portable fluorescence analyzer to detect the fluorescence value of the sample: Add the mixed solution to the microcalorimeter, make sure not to introduce bubbles into the sample, and gently tap the outside of the microcalorimeter to disperse the bubbles. Measure the fluorescence values of the sample and blank control using 1 × TE buffer as a blank; Corresponding to the concentration of standard solution (ng/ml)Perform linear regression on fluorescence intensity and prepare a standard curve.5). Measure the fluorescence value of the remaining samples. The fluorescence meter will provide a direct concentration reading, which can be used to generate a standard curve of DNA concentration. Final concentration of DNA to be tested Fluorescence reading value (ng/ml) / 100 6210 50 3195 40 2507 20 1261 10 620.8 5 298 4 258.8 2 152 0.5 43.8 0 0.72... 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 | 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 contentG665801Component100 TStorageG665801A2×GoldStar Probe One Step Buffer1.4 mL-20℃. Avoid freeze/ Thaw cycle. Protect from light.G665801BGoldStar Probe One Step EnzymeMix100 µL-20℃. Avoid freeze/ Thaw cycle. Protect from light.G665801C50×High ROX50 µL-20&#Product contentG665801Component100 TStorageG665801A2×GoldStar Probe One Step Buffer1.4 mL-20℃. Avoid freeze/ Thaw cycle. Protect from light.G665801BGoldStar Probe One Step EnzymeMix100 µL-20℃. Avoid freeze/ Thaw cycle. Protect from light.G665801C50×High ROX50 µL-20℃. Avoid freeze/ Thaw cycle. Protect from light.G665801DRNase-Free Water1.5 mL-20℃. Avoid freeze/ Thaw cycle. Product Introduction This product is a specialized kit for one-step Real-Time RT-qPCR using the probe method (TaqMan, Molecular Beacon, etc.). When using this product for Real Time RT-qPCR reaction, reverse transcription and quantitative PCR are carried out in the same reaction system, and there is no need to add reagents or open the cap of the tube during the reaction process, which avoids contamination and improves the experimental efficiency at the same time. With high detection sensitivity, strong fluorescence signal and high signal-to-noise ratio, this product is very suitable for the detection of RNA viruses and other trace RNA. The special buffer system contained in this product can maximize the effectiveness of reverse transcriptase and DNA polymerase at the same time and improve the efficiency of the reaction. A wider linear range can be obtained with this product, more accurate quantification of the target gene, good reproducibility and high confidence.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 (G665836) Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96 and others. Instruments that require High ROX calibration (G665801) ABI Prism 7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, and others.matters needing attention1.Before using the reagents in this kit, please mix them gently by turning them up and down to avoid foaming as much as possible, and use them after brief centrifugation.2.This product uses RNA as the template for one-step RT-PCR experiment, RNase contamination should be avoided during operation, it is recommended to operate RNA in a special area, use special instruments and consumables, the operator with a mask and disposable gloves and often change the gloves, the experiment-related consumables should be processed with 0.1% DEPC (diethyl ether pyrocarbonate) aqueous solution for 12 hours at 37℃, and autoclaved for 30 minutes before use. The consumables should be treated with 0.1% DEPC (diethylpyrocarbonate) aqueous solution at 37℃ for 12 hours and autoclaved for 30 minutes.3.Repeated freezing and thawing of each reagent in this kit should be avoided as much as possible; repeated freezing and thawing may degrade the product performance.4.This kit must use specific primers, the choice of primers can be selected according to specific experiments, the good or bad primer design directly affects the results of RT-qPCR reaction, the design of primers need to consider the GC content, primer length, primer position, the secondary structure of the PCR product and other factors, it is recommended to use a professional primer design software for design.5.This kit is recommended to use specific probes, and it is recommended to use professional design software for designing.UsageThe following examples are conventional reaction systems and conditions, which should be improved and optimized according to the different templates, primer structures and target fragment sizes in actual operation. (Please prepare the reaction solution on ice.)1. Dissolve RNA template, primers, 2× GoldStar Probe One Step Buffer, GoldStar Probe One Step EnzymeMix and RNase-Free Water and set aside on ice.2. PCR reaction system:reagents25µl reaction systemfinal concentration2×GoldStar Probe One Step Buffer12.5µl1×Forward Primer, 10µM0.5µl0.2µM¹⁾Reverse Primer, 10µM0.5µl0.2µM¹⁾Probe, 10µM0.5µl0.2µM²⁾GoldStar Probe One Step EnzymeMix1.0µl RNA TemplateXµl10pg-100ng³⁾50 x Low ROX or High ROX (optional)⁴⁾0.5µl1×RNase-Free WaterUp to 25µlNote: 1) Usually, better results can be obtained with a primer concentration of 0.2 µM, and 0.1-1.0 µM can be used as a reference for setting the range.(2) The concentration of the probe used is related to the fluorescence quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, please refer to the instrument manual or the specific requirements for the use of each fluorescent probe for the adjustment of the concentration in actual use.(3) Usually the amount of RNA template is 10pg-100ng as a reference. Since the templates of different species contain different copy numbers of target genes, the templates can be diluted in gradient to determine the optimal amount of template to use.(4) The excitation optical system varies from instrument to instrument, choose to add 50×Low ROX or 50×High ROX according to the instrument using fluorescence quantification.3. Mix well, centrifuge briefly, and collect the solution at the bottom of the tube.4.RT-PCR reaction conditions:Note: 1) The hot start enzyme used in this product must be activated under the condition of pre-denaturation 95℃, 5-10min. 2) It is recommended to use the two-step PCR reaction program, if you can not get good experimental results due to the use of primers with lower Tm value, etc., you can try to carry out the three-step PCR amplification, and the annealing temperature should be set in the range of 56℃-64℃ as a reference... 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 |