| Description | Inquire | Products content Box 1: Circularization reagentC666001Component16 TStorageC666001ASplint Oligo20 µL-20℃.Avoid freeze/thaw cycle. C666001B5×Splint Buffer T4250 µL-20℃.Avoid freeze/thaw cycle. C666001CDNA Ligase50 µL-20℃.Avoid freeze/thaw cycle. C666001DDigestion Products content Box 1: Circularization reagentC666001Component16 TStorageC666001ASplint Oligo20 µL-20℃.Avoid freeze/thaw cycle. C666001B5×Splint Buffer T4250 µL-20℃.Avoid freeze/thaw cycle. C666001CDNA Ligase50 µL-20℃.Avoid freeze/thaw cycle. C666001DDigestion Buffer20 µL-20℃.Avoid freeze/thaw cycle. C666001EDigestion Enzyme I70 µL-20℃.Avoid freeze/thaw cycle. C666001FDigestion Enzyme III25 µL-20℃.Avoid freeze/thaw cycle. Box 2: Magnetic Beads for DNA Purification and RecoveryC666001Component16 TStorageC666001GCMPure4×1.5 mL2-8℃Products IntroductionThe Cyclization Kit is a modular kit tailored for the MGI high-throughput sequencing platform. With this kit, PCR products after junction ligation can be prepared into single-stranded circular DNA libraries suitable for MGI sequencers. All reagents provided in the kit have been subjected to stringent quality control and functional validation to maximize the stability and reproducibility of library construction. Provide your own instruments, reagents and consumables1. Magnetic frame: DynaMagTM-2 (Cat. No. 12321D) is recommended.2. "Qubit" 3.0 Fluorescence Quantimeter (ThermoFisher, Cat. No. Q33216)3. Qubit" ssDNA Assay Kit (Invitrogen, Cat. No. Q10212)4. Anhydrous ethanol, EB (10 mM Tris-HCl, pH 8.0), NF Water (pH between 7.0 and 8.0).5. reaction tubes: low adsorption PCR tubes with 1.5 mIEP tubes are recommended: 5.Tip: It is recommended to use a high quality filter tip to prevent contamination of kits and libraries. Pre-experiment Preparation and Important Notes 1. Sample preparation.PCR product: 2330 ng total (total amount when multiple PCR products are mixed) in a volume of 49 pL (if the volume of PCR product is insufficient, add NF Water to bring the total volume to 49 pl). -PCR product: Fragment size: The fragment peak is between 200-500 bp. -PCR product fragment size: Fragment peaks between 200-500 bp. -PCR product modification: Fixed sequences (with Index) for MGISEQ-2000, MGISEQ-200 and BGISEQ-500 sequencing platforms were added.2. Reagent preparation-Remove the corresponding reagents from the kit, centrifuge briefly, and place the enzyme mixture on ice until ready to use: buffers need to be dissolved at room temperature before use, then centrifuged with shaking and placed on ice until ready to use, and NF Water and EB are placed at room temperature until ready to use: "Please make up the mixture on ice:Precipitation may appear after the buffer in the kit is dissolved, the precipitation does not affect the function of the reagent, please shake and mix well until the precipitation disappears and then use. Schematic diagram of the cyclization process procedurecyclize 1. 1 wl of Splint Oligo was added to the 49JI PCR product. The product was denatured and incubated on a PCR instrument at 95°C for 3 min, then immediately transferred to an ice bath and allowed to stand for 2 min. 2. The reaction mixture was prepared on ice according to the following system. 3. Add 15ul of the above reaction mixture to 50µl of denatured DNA.4. Place the above PCR tubes on the PCR instrument under the following conditions Reaction. digest 1. Prepare the digestion reaction solution on ice according to the following system. 2. After the cyclization reaction, add 8l of digestion reaction solution directly to the cyclization system, mix well, centrifuge briefly and then place the PCR tube on the PCR instrument and react under the following conditions. 3. Purification was carried out immediately after the reaction.Purification of digestive products1. Remove CMPure at room temperature 30 minutes prior to use and mix well with shaking.2. Transfer the digested product to a 1.5 mIEP tube, pipette 340 pICMPure into the digested product, mix well by gently blowing 10 times with a pipette and incubate for 10 minutes at room temperature.3. Instantaneous centrifugation, place the EP tube on a magnetic rack and let stand for 5 minutes until the liquid is clear, pipette and discard the supernatant.4. Keep the EP tube fixed on a magnetic rack, add 250ul of freshly prepared 80% ethanol, let it stand at room temperature for 1 minute, then carefully discard the supernatant.5. Repeat step 4 once, try to suck up the liquid at the bottom of the tube: Note: Do not suck up the magnetic beads, so as not to affect the yield.6. Keep the EP tube fixed on the magnetic rack, open the cap and dry it at room temperature for 5-10 minutes.7. Remove the EP tube from the magnetic rack, add 35ul of EB or NF Water for DNA elution, pipette blow to mix and dissolve at room temperature for 10 min.8. Centrifuge instantaneously, place the EP tube on a magnetic rack and let stand for 2 minutes until the liquid is clarified, transfer the supernatant to a new EP tube. -Store at 20C and leave to prepare DNB... Read More | Product introduction：Griess reagent can be used for spectrophotometric detection of nitrite. The reagent contains two chemicals, sulfonic acid and n- (1-naphthyl) ethylenediamine. Under acidic conditions, sulfamic acid is converted into diazonium salt by nitrite, which can form a highly Product introduction：Griess reagent can be used for spectrophotometric detection of nitrite. The reagent contains two chemicals, sulfonic acid and n- (1-naphthyl) ethylenediamine. Under acidic conditions, sulfamic acid is converted into diazonium salt by nitrite, which can form a highly colored azo dye with n- (1-naphthyl) ethylenediamine. This dye can be detected at 548 nm: because no is extremely unstable, it is oxidized to form nitrite and nitrate. Griess indirectly reflects the content of no by detecting the content of nitrite.Matters needing attention：1. before using Griess reagent, return it to room temperature and check the solution for precipitation. If Griess reagent I contains sediment when taken out, it can be placed in a 37 ℃ water bath until the sediment dissolves. 2. this product is potentially harmful. Avoid prolonged or repeated exposure. Avoid entering eyes, skin or clothing. Please wear lab clothes and disposable gloves for operation.Scope of application：No detectionComponent：Instruction：1.Griess Reagent I and II were taken out to restore the room temperature.2.Standard dilution : The standard NaNO2 ( 1-100 µM ) was diluted with the solution used for the sample to be tested. The standard was diluted to 1 µM, 10 µM, 20 µM, 40 µM, 80 µM and 100 µM, and 100 µL standard was added to each well. If the sample concentration is too low, the range of the standard curve can be appropriately reduced ( 1 µM, 2 µM, 3 µM, 4 µM, 6 µM, 8 µM, 10 µM ).3.Sample detection :( 1 ) According to the total volume of 200 µL / hole, 100 µL / hole sample was added to the 96-well plate ; if the sample is the supernatant of the culture medium, it can be sampled directly, and if there is sediment, the supernatant should be taken after centrifugation. If the sample is a cell or tissue, it can be quickly lysed by freeze-thaw, and then centrifuged to obtain the supernatant. The volume of less than 100 µL can be diluted with diH2O or 0.9 % NaCl ( corresponding standards also need to be diluted with diH2O or 0.9 % NaCl ).( 2 ) According to 50 µL / hole, Griess Reagent I was added to each hole.( 3 ) According to 50 µL / hole, Griess Reagent II was added to each hole.( 4 ) The absorbance was measured at 540 nm. If there is no 540 nm filter, 520-560 nm filter can also be. If there is no microplate reader or a suitable filter, the concentration of nitric oxide in the sample can also be determined by visual colorimetry. A more precise concentration gradient is required for the standard when visual colorimetric... 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 | Product content： Component O66550510 preps O665505 50 preps Blocking Buffer 100 ml 500 ml Antibody Pretreat Solution（ HRP/Mouse ） 1 ml 5 × 1 ml Dilution Buffer 100 ml 500 ml Wash Buffer（ 10× ) 100 ml 500 mlProduct Introduction:The one-step rapid WB assay kit (Product content： Component O66550510 preps O665505 50 preps Blocking Buffer 100 ml 500 ml Antibody Pretreat Solution（ HRP/Mouse ） 1 ml 5 × 1 ml Dilution Buffer 100 ml 500 ml Wash Buffer（ 10× ) 100 ml 500 mlProduct Introduction:The one-step rapid WB assay kit (mouse) is the latest Western Blot detection kit developed by Kangwei Century, which can obtain high-quality Western Blot results in about 1 hour. It is easy to operate, has high detection sensitivity, low background, does not require the addition of secondary antibodies, and has strong system stability. The conventional Western Blot indirect detection process (blocking, primary antibody binding, and secondary antibody binding) requires a long time, a complex experimental process, and requires multi-step condition optimization. After transferring the protein on the gel to the carrier membrane, incubate it with the blocking solution in the reagent kit for 5 minutes, and then incubate the carrier membrane with the primary antibody treated with antibody reaction solution. After washing three times (5 minutes each time), luminescence or colorimetric detection can be performed. This reagent kit is designed for use in experimental systems where the target protein primary antibody is derived from mice.Notes:1. The customer prepares their own mouse source primary antibody.2. Before using Blocking Buffer blocking solution, Antibody Pretreat Solution (HRP/Mouse) antibody reaction solution (mouse), and Wash Buffer (10 x) rinse solution, please mix thoroughly.3. If there is precipitation in the rinsing solution when stored at 2-8 ℃, please restore it to room temperature, dissolve the precipitation, and use it normally. The 1x rinsing solution can be stored at room temperature for one month.4. It is recommended to stain the membrane with reagents such as spring red after the transfer is completed, and cut off any excess parts on the membrane to increase the efficiency of the reagents.5. The optimal dilution amount for primary antibody and antibody reaction solution HRP (mouse) needs to be determined through preliminary experiments.6. The antibody reaction solution HRP (mouse), antibody dilution solution, and antibody dosage can be increased or decreased proportionally according to the size of the membrane.7. The antibody dilution solution containing the first antibody can be recycled and reused once. It is recommended not to reuse antibodies with poor specificity and affinity. If the recovered antibody is used within 1-2 days and stored at 2-8 ℃ for long-term storage, please freeze it at -20 ℃ to avoid repeated freeze-thaw cycles.8. If there is a high background, please adjust the amount of antibodies and increase the number of times the film is washed.9. All reagents in the reagent kit should be stored at 2-8 ℃ to avoid freezing and thawing.Operation steps:This product is suitable for the sealing and antibody incubation steps after membrane transfer, taking a 5 cm x 8 cm membrane as an example:1. Preparation of rinsing solution: Dilute 10 ml of Wash Buffer (10 x) with distilled water to 100 ml, which is 1 x Wash Buffer. Set aside. Use 8-10 ml for each film wash.2. Sealing: After the membrane transfer is completed, immerse the membrane in 10 ml Blocking Buffer and seal at room temperature for 5 minutes.3. Rinse: Pour off the sealing solution, add 8-10 ml of 1 x Wash Buffer, and rinse at a high speed on a shaker for 1 minute.4. Prepare antibody incubation solution while washing the membrane: Take Antibody Pretreat Solution (HRP/Mouse) 100 µ Add mouse derived primary antibody 3-10 into the centrifuge tube µ g. Suck and beat the gun head until thoroughly mixed, and incubate at room temperature for 5 minutes. Add to 10 ml Dilution Buffer and mix well. Note: 1) The dosage of primary antibody can also be adjusted according to the dilution of the antibody. Taking the final dilution of antibodies at 1:1000 as an example, take 100 µ Add HRP (mouse) antibody reaction solution into the EP tube and add 10 µ Add the first antibody to 10 ml of antibody diluent, mix well, and incubate at room temperature for 5 minutes. 2) If the membrane area is small, the amount of antibodies, reaction solution, and diluent can be reduced proportionally.5. After completing step 3, pour out the rinsing solution and add the antibody incubation solution mixed with primary antibody, Antibody Pretreat Solution (HRP/Mouse), and Dilution Buffer to the membrane (ensuring that the incubation solution completely submerges the surface of the membrane). Incubate at room temperature on a shaker at around 60 rpm for 40 minutes.6. Discard (recover) the antibody incubation solution and rinse 3-5 times with the prepared 1 x Wash Buffer, each time for 3 minutes. 7. Conduct subsequent testing. It is recommended to use ECL or DAB methods for testing.Application examples:Example 1 Antigen is 293T cell lysateA: Normal WB control: beta actin mouse monoclonal antibody (CW0096) 5 µ Incubate at room temperature for 40 minutes, wash the film and dilute the secondary antibody sheep anti mouse HRP (CW0102) 1:10000. Incubate at room temperature for 40 minutes and expose ECL (CW0049).B: One step method WB: beta actin mouse monoclonal antibody (CW0096) 5 µ Incubate at room temperature for 40 minutes and expose ECL (CW0049).Example 2 Antigen is E. coli multi label protein lysateC: Normal WB control: GST mouse monoclonal antibody (CW0084) 2.5 µ Incubate at room temperature for 40 minutes, wash the film and dilute the secondary antibody sheep anti mouse HRP (CW0102) 1:10000. Incubate at room temperature for 40 minutes and expose ECL (CW0049).D: One step method WB: GST mouse monoclonal antibody (CW0084) was incubated at room temperature with 2.5ug for 40 minutes, and ECL (CW0049) was exposed... Read More |