| 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 | B665530 Component 50 T 200 T Storage B665530A Buffer RCL 125 mL 2×260 mL 2-8℃ B665530B Buffer GR 15 mL 50 mL RT B665530C Buffer GL 15 mL 50 mL RT B665530D Buffer GW1 (concentrate) 13 mL 52 mL RT B665530E Buffer GW2 (concentrate) 15 mL 50 mL RT B665530F Buffer GE 15 mL 60 mL RT B665530G B665530 Component 50 T 200 T Storage B665530A Buffer RCL 125 mL 2×260 mL 2-8℃ B665530B Buffer GR 15 mL 50 mL RT B665530C Buffer GL 15 mL 50 mL RT B665530D Buffer GW1 (concentrate) 13 mL 52 mL RT B665530E Buffer GW2 (concentrate) 15 mL 50 mL RT B665530F Buffer GE 15 mL 60 mL RT B665530G Proteinase K 1.25 mL 4×1.25 mL RT B665530H Spin Columns DM with Collection Tubes 50 sets 200 sets RTProduct IntroductionThis reagent kit is suitable for extracting total DNA, including genomic DNA, mitochondrial DNA, and viral DNA, from fresh or frozen whole blood (blood samples treated with anticoagulants such as citrate, EDTA, or heparin), plasma, serum, erythrocyte sedimentation rate brown layer, lymphocytes, cell-free body fluids, and other samples. This product can process 0.1-1 mL of whole blood with a maximum yield of 30% µ g. It can purify DNA with sizes ranging from 100 bp to 50 kb. The purified DNA has high yield and good quality, and can remove protein, pigment, lipid, and other inhibitory impurities to the maximum extent. It can be directly used for PCR, fluorescence quantitative PCR, enzyme digestion, and Southern Blot experiments.Self prepared reagent: anhydrous ethanol.Preparation and important precautions before the experiment:1. The sample should avoid repeated freeze-thaw cycles, otherwise it may result in smaller extracted DNA fragments and a decrease in extraction volume.2. This reagent kit can extract up to 0.1-1 mL of whole blood samples or 1 × 107 white blood cells.3.Before the first use, anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the instructions on the reagent bottle label.4. Before use, please check if there is any crystallization or precipitation in the Buffer GL. If there is any crystallization or precipitation, please incubate the Buffer GL in a 56 ℃ water bath and dissolve it again.5. The Buffer RCL in the reagent kit cannot be used again after being turbid.Operation steps:1. Sample processing: 1a When extracting 200 uL of blood sample, add the sample to the centrifuge tube (provided) and proceed directly to the next step of the experiment. 1b When the blood sample size is less than 200 µ When L, add Buffer GR to make up for 200 µ L. Proceed to the next step of the experiment. 1c When the blood sample size exceeds 200 µ When L is reached, add 1-2 times the volume of Buffer RCL, gently vortex or invert and mix well. Centrifuge at 12000 rpm (~13400 × g) for 1 minute and carefully discard the supernatant. If there is still red in the sediment, repeat the above steps once. Then add 200 to the precipitate µ Shake the buffer GR until thoroughly mixed before proceeding to the next step of the experiment. 1d If the processed blood sample is anticoagulant from poultry, birds, amphibians, or lower level organisms, its red blood cells are nucleated cells, and the blood sample size is 5-20 µ L. Can be added to Buffer GR to make up to 200 µ Follow up experiments will be conducted afterwards. Note: If downstream experiments are sensitive to RNA, 4 can be added µ L RNase A (100mg/mL) solution, shake for 15 seconds, and leave at room temperature for 5 minutes. RNase A reagent kit is not provided. If needed, you can order it separately from our company, item number: CW0601S.2. Add 20 to the above solution µ L Protein K, mix well.3. Add 200 µ Shake with L Buffer GL until thoroughly mixed. Note: Do not pre mix Protein K and Buffer GL.4.Incubate at 4.56 ℃ for 10 minutes, invert and mix several times during this time. Attention: The DNA production has reached its maximum after 10 minutes of incubation, and further extension of incubation time has no effect on DNA production and purity.5. Add 200 µ L anhydrous ethanol, invert and mix several times. Short centrifugation causes the liquid on the tube wall and wall cover to concentrate at the bottom of the tube.6. Add all the solution 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 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 L Buffer GW1 (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. Note: If the extracted sample is the blood genome of species such as mice or monkeys that are difficult to remove heme, it is recommended to repeat step 7.8. Add 500 to the adsorption column µ L Buffer GW2 (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. Note: To further improve DNA purity, repeat step 8.9.Centrifuge at 9.12000 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.)10. Place the adsorption column in a new centrifuge tube (provided by oneself) and add 50-200 to the middle of the adsorption column in the air 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 is not high. 2) If the final concentration of DNA needs to be increased, the obtained DNA eluent can be added back to the adsorption membrane, left at room temperature for 2-5 minutes, and centrifuged at 12000 rpm for 1 minute. 3) Because DNA stored in water is affected by acidic hydrolysis, if long-term storage is required, it is recommended to elute with Buffer GE and store at -20 ℃... Read More | 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 | Products contentNote: The amount of individual primers used is 1 µl, each N7-end primer can perform 10 DNA library constructs, and each kit can perform 240 DNA library constructs.Products IntroductionThis kit is a companion kit to the transposase-based Rapid DNA Library Construction Kit for Products contentNote: The amount of individual primers used is 1 µl, each N7-end primer can perform 10 DNA library constructs, and each kit can perform 240 DNA library constructs.Products IntroductionThis kit is a companion kit to the transposase-based Rapid DNA Library Construction Kit for Illumina platform library construction. Each kit contains one N5 primer and 24 N7 primers, which can be used to prepare 24 different single-ended Index libraries. All reagents provided in the kits have been subjected to stringent quality control and functional validation to maximize the stability and reproducibility of library construction. The libraries can be used for sequencing on Illumina platforms such as HiSeq X-10/4000/2500/2000 and MiSeq. Provide your own instruments, reagents and consumables1. Magnetic frame: DynaMagTM-2 is recommended.2. DNA purification and recovery kit: It is recommended to use Kangwei DNA purification and recovery kit by magnetic bead method.3. DNA building kit: It is recommended to use the Kangwei Century transposase method second-generation sequencing rapid DNA building kit.4. Anhydrous ethanol.5. Reaction tubes: It is recommended to use low adsorption PCR tubes with 1.5 ml centrifuge tubes; Tip: It is recommended to use a high quality filter tip to prevent contamination of kits and library samples. Pre-experiment Preparation and Important NotesPlease centrifuge briefly before opening the cap so that the liquid collects at the bottom of the tube to avoid cross-contamination between different primers. procedureFor the use of the CombiVision Second Generation Sequencing Multisample Primer Kit, please follow the CombiVision Second Generation Sequencing Rapid DNA Library Kit protocol. Index N501 Primer for Illumina Index N901-N996 Primer for Illumina... Read More | Inquire |