| 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 | Product Descriptionalpha-L-fucoside fucohydrolase, alpha-L-fucosidase, alpha-(1-3,4) fucosidaseAlpha (1-3,4) Fucosidase The enzyme is very efficient and recognises α1-3,4 fucosylated glycans (e.g. Lewis X/A epitopes, including their sialylated counterparts) and hydrolyses terminal α1-3 andProduct Descriptionalpha-L-fucoside fucohydrolase, alpha-L-fucosidase, alpha-(1-3,4) fucosidaseAlpha (1-3,4) Fucosidase The enzyme is very efficient and recognises α1-3,4 fucosylated glycans (e.g. Lewis X/A epitopes, including their sialylated counterparts) and hydrolyses terminal α1-3 and α1-4 fucosyl linkages in these substrates without the need to remove sialic acid moieties.For removing core fucose linked α-(1-6) to the core GlcNAc of a GlcNAc-GlcNAc disaccharide structure we recommend our Alpha-(1-6) Fucosidase.• Non-sialidase dependant hydrolysis of antennary fucose moieties• Effective on both glycopeptides and free glycans• Highly specific (α1-3,4 fucosylated glycans)• Kit includes enzyme plus reaction buffer.• Sufficient for up to 50 samplesα(1-3,4) Fucosidase is useful for:nbsp;nbsp;Fucose linkage determinationnbsp;nbsp;Deglycosylating glycoproteins with Lewis structuresContentsAlpha-(1-3,4)-Fucosidase – 200 mM citrate buffer pH 6 containing 250 mM NaCl5x Reaction Buffer – 250 mM sodium phosphate pH 6... Read More | Inquire | 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 | Cell viability and cytotoxicity assays are usually used for drug screening and compound cytotoxicity testing. The CCK-8 kit uses highly water-soluble tetrazolium salt ( called WST-8 ) to produce water-soluble WST-8 for cell proliferation and cytotoxicity assays. Unlike MTT, WST-8 and WST-8 have no Cell viability and cytotoxicity assays are usually used for drug screening and compound cytotoxicity testing. The CCK-8 kit uses highly water-soluble tetrazolium salt ( called WST-8 ) to produce water-soluble WST-8 for cell proliferation and cytotoxicity assays. Unlike MTT, WST-8 and WST-8 have no cytotoxicity in cell culture medium, so multiple downstream experiments can be performed using the same detection plate. CCK-8 method is a convenient colorimetric method for the determination of cell viability. It does not need the solubilization process and only needs the least steps to provide the results. The CCK-8 method can be used for the determination of 96-well microplates and high-throughput screening of 384-well microplates. Advantage：At present, the commercially available liquid CCK-8 kits generally have defects such as harsh storage conditions ( -4C or -20 ), unstable use in different pH ranges, and easy deterioration ( discoloration or precipitation ). The solid instant CCK-8 kit adopts a new formula and Swiss process, which overcomes these shortcomings of the liquid CCK-8 kit. It can be stored at room temperature for a long time ( > 3 years ), ready to use, stable in a wide pH range, and the experimental results are more reliable. Compared with the liquid CCK-8 kit, the solid-soluble CCK-8 kit has higher sensitivity and the biological response time is shortened by half.Application scope:It can be used for drug screening, cell proliferation assay, cytotoxicity assay, tumor drug sensitivity test and activity detection of biological factors. Operating instructions:This reagent kit can be used for drug screening, cell proliferation assay, cytotoxicity assay, tumor drug sensitivity assay, and activity detection of biological factors.1. Carefully and slowly tear along the gap in the packaging bag;2. Pour all the powder in the bag into a clean container containing 10mL of ultrapure water, shake continuously for 1 minute, and use it when the solid is completely dissolved;3. Unused reagents must be stored at low temperatures below 4 ℃.Equipment required for testing:Enzyme reader 96 well plate with 450-490 nm filter;Carbon dioxide incubator;96 well plate, sterilized transparent plate for cell detection;Multi channel pipette (8 or 12 channels: 10-100 µ l);Blood cell counter or cell counter.Cell viability testing:1. Inoculate cell suspension (100 µ l/well) into a 96 well plate and pre culture the plate in a carbon dioxide incubator for 24 hours (37 ℃, 5% CO2);2. Add 10 µ l of CCK-8 solution to each well (be careful not to generate bubbles in the well as it may affect the reading of OD value);3. Incubate the culture plate in the incubator for 1-4 hours;4. Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader;5. If the OD value is not determined temporarily, 10 µ l of 0.1M HCI solution or 1% w/v SDS solution can be added to each well, and the culture plate can be covered and stored in the dark at room temperature. Within 24 hours of measurement, the absorbance will not change.Cell proliferation toxicity testing:1. Inoculate cell suspension (100 µ l/well) into a 96 well plate and pre culture the plate in an incubator for 24 hours (37 ℃, 5% CO2);2. Add 10ul of different concentrations of the substance to be tested to the culture plate;3. Incubate the culture plate in the incubator for an appropriate period of time (e.g. 6, 12, 24, or 48 hours);4. Add 10 µ l of CCK-8 solution to each well (be careful not to generate bubbles in the well as they may affect the reading of the OD value);5. Incubate the culture plate in the incubator for 1-4 hours;6. Measure the absorbance at 450nm using an enzyme-linked immunosorbent assay (ELISA) reader;7. If the OD value is not determined temporarily, 10 µ l of 0.1M HCI solution or 1% w/v SDS solution can be added to each well, and the culture plate can be covered and stored in the dark at room temperature. Within 24 hours of measurement, the absorbance will not change.Calculation method for cell survival rate/inhibition rate:Cell survival rate=[As Ab)/(Ac Ab)] x 100%Inhibition rate=[(Ac As)/(Ac Ab)] x 100%As: absorbance of experimental wells (including cells, culture medium, CCK-8 solution, and drug solution);Ac: absorbance of control wells (including cells, culture medium, CCK-8 solution, without drugs);Ab: Blank well absorbance (including culture medium and CCK-8 solution, excluding cells and drugs).Points for attention： 1.Unused reagents must be stored at low temperature below 4 °C, and stored in the dark at-20 °C for two years after unpacking, so as to avoid repeated thawing ; 2.The culture time of CCK-8 is generally 1-4 hours, but the naked eye can be taken out to observe the color degree in about 30 minutes. According to the cell type, the conditions need to be explored. The best reaction time of CCK-8 is based on the best time of specific color development.3. It is recommended to do a few holes to explore the number of inoculated cells and the culture time after adding CCK-8 reagent ; 3.The WST-8 in this kit will react with reducing agents ( such as some antioxidants ) to interfere with the detection. Before the cell proliferation-toxicity test, the background OD can be checked to confirm whether there is a reducing agent in the substance to be tested. If the effect of reducing agent needs to be removed, the fresh medium can be replaced before adding CCK-8 ( remove the medium, wash the cells twice with the medium, and then add the new medium ) ; 4.Phenol red in the medium does not affect the experimental results, and the absorbance of phenol red can be eliminated by deducting the absorbance of the background in the blank hole during calculation, so it will not affect the detection. 5.It is recommended to use a multi-channel pipette to reduce the difference between parallel holes. When adding CCK-8 reagent, it is recommended to add it obliquely to the wall of the culture plate, not to insert it under the liquid surface of the medium, which is easy to produce bubbles and interfere with OD determination. 6.If the drug contains metal, it has an effect on the color of CCK-8. The final concentration of 1mM lead chloride, ferric chloride and copper sulfate will inhibit the color reaction of 5 %, 15 % and 90 %, and reduce the sensitivity. If the final concentration is 10mM, the color reaction will be 100 % inhibited ; 7.When using a 96-well plate for detection, if the cell culture time is long, attention should be paid to the evaporation problem. On the one hand, because a circle around the 96-well plate is the easiest to evaporate, the method of discarding the surrounding circle can be adopted, and the same amount of PBS, water or culture medium can be added. On the other hand, the 96-well plate can be placed near the water source in the incubator to alleviate evaporation ; 8.When using standard 96-well plates, the minimum inoculation amount of adherent cells is at least 1,000 cells / well ( 100µl medium ). The sensitivity of detecting white blood cells is relatively low, so it is recommended that the inoculation amount should not be less than 2,500 cells / well ( 100 µl medium ). If you want to use a 24-well plate or a 6-well plate experiment, first calculate the corresponding inoculation amount per well, and add the CCK-8 solution according to 10 % of the total volume of the medium per well ; 9.Cell culture time varies according to the type and number of cells ( per well ), usually the color of white blood cells is weak, requiring a longer culture time ( 4 hours ) and a large number of cells ( ~ 105 cells / well ) ; 10.CCK-8 reagent is very low toxic to cells. The continuous reaction between it and dehydrogenase in living cells makes the color of the solution deepen and the OD value increase. The following methods can terminate the CCK-8 reaction ( 96-well plate ) : a ) After the color reaction, the culture plate was placed in a refrigerator at 4 ° C ; b ) 10µL 0.1MHCL solution was added to each well ; c ) 10 µL 1 % ( w / v ) SDS ( sodium dodecyl sulfate ) solution was added to each well. After the reaction stopped, the OD value should be measured within 24 hours. 11.To determine the specific number of cells, it is recommended to do the standard curve at the same time... Read More |