| Description | 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 | Product introduction：This kit uses uniqcell lysis and heme / protein precipitation technology, combined with DNA preparation membrane to selectively adsorb DNA to achieve the purpose of purifying genomic DNA.Scope of application：Nucleic acid extraction and purification | Products contentN669983Component240 TStorageN669983AIndex N501 Primers for Illumina240 µL-20℃. Avoid freeze/thaw cycle.N669983BIndex N973-N996 Primers for Illumina24×10 µL-20℃. Avoid freeze/thaw cycle. Products IntroductionThis kit is a companion kit to the transposase-Products contentN669983Component240 TStorageN669983AIndex N501 Primers for Illumina240 µL-20℃. Avoid freeze/thaw cycle.N669983BIndex N973-N996 Primers for Illumina24×10 µL-20℃. Avoid freeze/thaw cycle. 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 DNA purification and recovery kit by magnetic bead method.3. DNA building kit: It is recommended to use the 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 IlluminaIndex N901-N996 Primer for Illumina... Read More | Products contentN665954Component24 T96 TStorageN665954ATPS V136 µL144 µL-20℃. Avoid freeze/thaw cycle.N665954B5×FA Reaction Buffer96 µL384 µL-20℃. Avoid freeze/thaw cycle.N665954CTS Buffer72 µL288 µL-20℃. Avoid freeze/thaw cycle.N665954D2× Products contentN665954Component24 T96 TStorageN665954ATPS V136 µL144 µL-20℃. Avoid freeze/thaw cycle.N665954B5×FA Reaction Buffer96 µL384 µL-20℃. Avoid freeze/thaw cycle.N665954CTS Buffer72 µL288 µL-20℃. Avoid freeze/thaw cycle.N665954D2× PCR Mix600 µL2×1.2 mL-20℃. Avoid freeze/thaw cycle. * This kit is suitable for human genomic DNA library construction, the starting template DNA input amount is 1 ng, our company also has 50 ng and 5 ng of human genomic DNA starting transposase method library construction kit, in order to get a higher quality library, different starting amount of DNA is recommended to use different kits. Products IntroductionThis kit is developed for Illumina's high-throughput sequencing platform and provides the enzyme premix system and reaction buffer for genomic DNA library construction, including all components except PCR primers. Compared with the traditional library construction kits, this kit adopts the new transposase method for library construction, which can complete DNA fragmentation, end repair and junction reaction in one simple enzymatic reaction, significantly reducing the amount of template, reducing the number of experimental steps, and shortening the time of library construction; it adopts the high-fidelity DNA polymerase for library enrichment, and the preference-free PCR amplification can expand the coverage area of the sequence, which can be used for efficient and effective sequencing. The use of high-fidelity DNA polymerase for library enrichment and preference-free PCR amplification broadens the coverage area of the sequence and enables efficient preparation of DNA libraries for Illumina's second-generation sequencing platform. The kit is suitable for use with 1 ng of starting template DNA, and all reagents in the kit have been subjected to stringent quality control and functional validation to maximize the stability and reproducibility of library construction.Product Features● DNA fragmentation and junction ligation in one step.● Ultra-fidelity amplification minimizes amplification preference.Provide your own instruments, kits 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. Library PCR primer kit: It is recommended to use Kangwei transposase method for second generation sequencing multi-sample primer kit.4. Anhydrous ethanol, deionized water (pH between 7.0 and 8.0).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 Notes1. Avoid repeated freezing and thawing of reagents.2. PCR products are easily contaminated due to improper operation, resulting in inaccurate results. It is recommended to isolate the PCR reaction system preparation area from the PCR product purification area, and to use special pipettes to clean the experimental areas at regular intervals.3. Bead purification: the beads should be equilibrated to room temperature before use, all operations on the beads should be carried out at room temperature, 80% ethanol should be dispensed freshly, the beads should be rinsed and dried until the surface is free of liquid reflections and has a frosted appearance, insufficient drying of the beads will cause ethanol residue that will affect the subsequent experiments, and over-drying of the beads will affect the efficiency of DNA recovery.4. The kit is suitable for human genomic DNA library construction, if the DNA sample is a PCR product, it should be ensured that its length>.500 bp, since transposases do not work on DNA ends, it is recommended to extend the PCR product by 50-100 bp at each end of the PCR product to avoid low coverage of the ends for sequencing. Sample PreparationDNA purity requirement: A260/A280 = 1.8-2.0. Sample DNA: dissolved in ultrapure water.DNA quantification: Too much or too little DNA will affect the quality of the library. It is recommended to use Nano to test the purity of the genomic DNA and then use Qubit to test the concentration of the genome (do not use any absorbance-based assay for template quantification). Schematic diagram of DNA banking process procedureDNA fragmentation, junction reaction 1. Add the following reagents to a 200 µl PCR tube:2. Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.3. Place the above PCR tubes in the PCR instrument with the hot cap on and program the reaction as follows:inactivation reactionAfter the DNA is fragmented, the enzyme is still in a high active state, so it should be removed from the PCR instrument immediately and terminated by adding the Reaction Termination Buffer, in order to prevent the DNA from being fragmented too much and resulting in smaller library fragments.1. Add 3 µl of TS Buffer to the PCR tube containing the fragmentation product.2. Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.3. Incubate at room temperature for 5 min, or if the room temperature is too low, place the reaction on a PCR instrument at 25°C with the thermal cover closed.PCR amplification1. Add the following reagents to a 200 µl PCR tube.2. Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube. 3. Place the above PCR tubes in the PCR instrument with the thermal cap open, and the reaction program is as follows:Selective recovery of library DNA fragmentsIt is recommended to use CombiVision Magnetic Beads DNA Purification and Recovery Kit for selective recovery of DNA fragments. When different sizes of DNA fragments are required, the amount of magnetic beads used is different, please refer to the attached table for the specific amount of magnetic beads used.(If using other brands of magnetic beads, you need to figure out the optimal amount of magnetic beads by yourself).Note: Amplification products can also be fragment length sorted and purified using the Gum Recovery Kit. If there is no special requirement for library length distribution, amplification products can also be purified directly from DNA fragments without selective recovery of DNA fragments as described on page 4 of the manual.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. Transfer the PCR products to a 1.5 ml centrifuge tube, rehydrate to 100 µl, add several volumes of magnetic beads equilibrated to room temperature, vortex for 5 seconds, and let stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant until the solution is clear, and carefully aspirate the supernatant and transfer it to a new 1.5 ml centrifuge tube.Note: Do not discard the top clear.4. Add several volumes of magnetic beads to the supernatant, vortex and shake for 5 seconds, then let stand at room temperature for 5 minutes.5. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant until the solution is clear, carefully aspirate the supernatant and discard it, avoiding contact with the beads that have bound the target DNA.Note: Do not discard the beads.6. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.7. Repeat step 6 once.8. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, add 20 µl of ddH2O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.9. Remove the tube from the magnetic rack, vortex to completely resuspend the beads, and allow to stand at room temperature for 5 minutes. Centrifuge briefly, place the tube on the magnetic rack until the solution is clear, and transfer the supernatant solution to a new tube. Table: Suggested amount of magnetic beads for different segment selection recoveryLibrary DNA fragment purificationWe recommend the use of the Kangwei Century Magnetic Bead Method DNA Purification and Recovery Kit.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. 50 µl of magnetic beads equilibrated to room temperature were added to the PCR product, vortexed and shaken for 5 seconds, and then left to stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant solution until the solution is clear (approximately 3-5 minutes), carefully aspirate the supernatant and discard it, avoiding contact with the beads that have bound the target DNA. Note: Do not discard the beads.4. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the centrifuge tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.5. Repeat step 4.6. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, add 25 µl of ddH2O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.7. Remove the tube from the magnetic rack, vortex to completely resuspend the beads, and allow to stand at room temperature for 5 minutes. Centrifuge briefly, place the tube on the magnetic rack until the solution is clear, and transfer the supernatant solution to a new tube. Library quality controlDetermination of library concentrationIn order to obtain high-quality sequencing results, accurate quantification of DNA libraries is required, and the first recommendation is to use Real-timePCR methods are used for absolute quantification of DNA libraries. Additionally, fluorescent dye methods such as the Qubit method or the fluorescent dye picogreen method can be used; do not use quantification methods based on absorbance measurements here. The following approximate formula can be used to convert the molar concentration of the DNA library.Library fragment distributionThe prepared DNA libraries can be detected by agarose gel electrophoresis or Agilent 2100 Bioanalyzer.Range of segment length distributions... Read More | Product introduction：Reporter gene detection is an important tool for analyzing the interaction between potential cis elements (such as promoters, enhancers and silencers) and trans acting factors in the flanking region of structural genes in the field of modern molecular biology. Firefly Product introduction：Reporter gene detection is an important tool for analyzing the interaction between potential cis elements (such as promoters, enhancers and silencers) and trans acting factors in the flanking region of structural genes in the field of modern molecular biology. Firefly luciferase is widely used in gene regulation and drug screening. Firefly luciferase is a protein with a molecular weight of about 61 KD. In the presence of ATP, magnesium ions and oxygen, it can catalyze the production of oxyluciferin from luciferin. In the process of luciferin oxidation, it will produce a light signal. The optical signal of this kit is a kind of instantaneous light, which needs to be detected immediately after adding the working solution. The half-life of optical signal is about 5 min.Instruction：1.Working fluid configuration ( 1 ) Restore all components to room temperature. ( 2 ) The component B ( stock solution ) was fully diluted with component A to prepare a 0.2 mg / mL firefly luciferase working solution, which was vortexed and shaken to ensure full mixing. Note : The firefly luciferase working solution cannot be repeatedly frozen and thawed. If the dosage of a single experiment is small, it is recommended to subpackage according to a single dosage. At room temperature, the activity decreased by about 10 % after the working solution was configured for 3 h, and the activity decreased by about 25 % after 5 h. 2.chemiluminescence value detection ( 1 ) The cell culture plate was taken out from the incubator and incubated at room temperature for 20 min to restore it to room temperature ( 22-25 ° C ). ( 2 ) Add the same volume of firefly luciferase working solution with the medium to the culture plate and mix well. ( 3 ) Incubation at room temperature for 5 min. Note : The incubation time can be adjusted according to cell type and cell number. ( 4 ) The values were read by multifunctional microplate reader or chemiluminescence instrument ( instrument parameters : the determination time was 10 s, the determination interval was 2 s ).Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. the strongest wavelength of bioluminescence catalyzed by firefly luciferase is 560 nm. 3. to prevent interference between holes, it is recommended to use white opaque orifice plate.Recommendation：Component B is recommended to use sterile water in advance to configure 2 mg / mL storage solution, A component and B component configured as storage solution, and small batch packaging according to the experimental requirements. The detection working fluid is recommended to be used now to avoid repeated freezing and thawing. Component：One-Step Firefly Luciferase Assay Buffer；D-Luciferin Scope of application：Mainly used for ADCC detection... Read More |