| Description | Inquire | Cell proliferation detection is a basic experimental method to evaluate the health of cells, genotoxicity and the effect of antitumor drugs. The most accurate method to detect cell proliferation is the BrdU method. Edu detection kit is a revolutionary breakthrough of BrdU method. Edu (5-Cell proliferation detection is a basic experimental method to evaluate the health of cells, genotoxicity and the effect of antitumor drugs. The most accurate method to detect cell proliferation is the BrdU method. Edu detection kit is a revolutionary breakthrough of BrdU method. Edu (5-ethynyl-2 '- deoxyuridine) is a pyrimidine analog that integrates into the DNA duplex during DNA synthesis. Edu detection is based on the "click" reaction. A copper catalyzed azide reacts covalently with alkynes to form covalent bonds. In this kit, edu contains alkynes, Aladdin ® 488 / 555/594/647a azide dyes contain azide compounds. The edu labeling proliferation of click method is rapid and effective, and easy to use. BrdU method requires DNA denaturation (such as acid denaturation, thermal denaturation or digestion with DNase) to expose BrdU, so as to facilitate BrdU antibody binding; The edu method only needs paraformaldehyde fixation and Triton X-100 penetration to make the detection reagent enter the cells, and only a small amount of azide dye is needed to label the integrated edu very effectively. This kit contains all components required for edu method detection, and can be used for proliferation detection of cultured cells in vitro.Component:Product parameters：555/565 nm；Instruction： Experimental materials (self provided). 10 mM PBS, pH 7.2-7.6. 4% paraformaldehyde fixing solution (in PBS)Propensive reagent (0.5% Triton X -100 in PBS). 2 mg/mL glycine solution (in ddH2O). 3% BSA in PBS, pH 7.2-7.6. 1% BSA in PBS, pH 7.2-7.6. ddH2O. 96/24/12/6 well culture plate or dishFluorescence microscopy detection method1. Cell cultureTake logarithmic growth stage cells and inoculate them into a 96 well plate with 4 × 103-1 × 105 cells per well (the number and density of cells can be adjusted according to cell size, growth rate, and specific requirements of experimental treatment), and culture until normal growth stage.2. Drug treatmentPerform various drug treatments according to experimental needs.3. EdU marking(1) Dilute EdU solution (component A) in a certain proportion with complete cell culture medium to an appropriate concentration, then add it to the cells and mix well; Set up a negative control group without EdU treatment.Note: The labeling concentration of EdU needs to be adjusted according to cell type, and it is recommended to explore with an initial concentration of 10 µ M. In the pre experiment, it is recommended to set an EdU concentration gradient, which can be referred to in Tables 2 and 3.(2) Incubate in a cell culture incubator for 2 hours.Note: The optimal incubation time is related to the cell cycle. Most tumor cell lines can use a 2-hour incubation time, as shown in Appendix 2. The concentration of EdU is related to the incubation time, and high concentrations, such as 10-50, should be used for short-term incubation (<2 hours) µ M; Long term incubation (>24 hours) should use low concentrations, such as 1-10 µ M; You can also refer to Appendix 3.4. Cell fixation and permeation promotionNote: For experiments that require cell surface antigen labeling, it can be considered to wash the cells twice with a 3% BSA washing solution after completing EdU incubation, before cell fixation and permeation promotion.(1) After incubation, remove the culture medium. Wash cells twice with 1X PBS for 5 minutes each time to remove EdU residues that have not been incorporated into DNA. Cells with weak adhesion can reduce cleaning intensity. Join 50 µ After incubating at room temperature for 20 minutes with 4% paraformaldehyde fixative, remove the fixative.(2) Add 50 to each hole µ L 2 mg/mL glycine solution, incubate at room temperature for 5 minutes, and neutralize the remaining fixed solution.(3) At a rate of 100 per hole µ Wash cells twice with 3% BSA.(4) Remove the washing solution and add 100 µ L 0.5% Triton X -100, incubate at room temperature for 10 minutes.5. EdU detectionNote: Each sample in this reference step uses 100 µ The working fluid of L can be adjusted by users according to their own sample situation.(1) Prepare 1 x Click iT EdU reaction buffer (component C): Dilute component C 10 times with ddH2O.(2) Configure 5 x Click iT EdU buffer additives (component E): add 300 µ Mix L of ddH2O into a 30 mg E component tube (final concentration of 100 mg/mL) until completely dissolved. After use, the remaining storage solution is stored at -20 ℃ and can be stored for one year. Once the solution turns brown, it indicates that the active ingredients have degraded and cannot be reused.Note: Different specifications of component E are dissolved in ddH2O according to this ratio, and prepared into a 5 x storage solution for future use.(3) Prepare 1 x Click iT EdU buffer additive: Dilute 5 x Click iT EdU buffer additive with ddH2O to 1 x, and the solution should be prepared and used immediately.(4) Prepare Click it working solution according to Table 1.Table 1 Click it working fluidReaction componentsTaking the sample size of 10 holes as an example1×Click-iT EdU Reaction buffer855 µLCuSO4 (Component D)40 µLYF® 488/555/594/647A Azide(Component B)5 µL1×Click-iT EdU Buffer additives100 µLTotal volume1 mL(5) Remove penetration enhancer, 100 per well µ Wash twice with 3% BSA washing solution of L.(6) Add 100 to each hole µ L Click iT working solution, evenly covering cells.(7) Incubate at room temperature in dark for 30 minutes.(8) Remove Click-iT working fluid and add 100 µ After washing cells twice with 3% BSA, remove the washing solution and add 100 µ L PBS keeps cells moist. If there are no other special requirements, photography analysis can be carried out.6. DNA re staining (optional)(1) Using 100 µ Wash the cells once with PBS and remove the washing solution.(2) Dilute Hoechst 33342 (component F) 2000 times with PBS.(3) Add 100 to each hole µ Incubate L 1 x Hoechst 33342 solution at room temperature in dark for 15-30 minutes.(4) Remove Hoechst 33342 solution and use 100 µ Wash cells twice with PBS.7. Imaging and analysisIt is recommended to take fluorescence microscopy photos immediately after staining is completed for observation; If conditions permit, please store in a dark and moist environment at 4 ° C for 3 days before taking photos. Flow cytometry detection method1. Cell cultureInoculate 1 × 105~3 × 106 cells per well into a 6-well plate.2. Drug treatmentPerform various drug treatments according to experimental needs.3. EdU labeled cells(1) Dilute EdU solution (component A) in a certain proportion with complete cell culture medium to an appropriate concentration, then add it to the cells and mix well; Set up a negative control group without EdU treatment.Note: The labeling concentration of EdU needs to be adjusted according to cell type, and it is recommended to explore with an initial concentration of 10 µ M. In the pre experiment, it is recommended to set an EdU concentration gradient, which can be referred to in Tables 2 and 3.(2) Incubate in a cell culture incubator for 2 hours. The time of EdU incubation of cells can be directly used as an indicator for measuring cell DNA synthesis, and the choice of time point and incubation time depend on the cell growth rate. Pulse labeled cells incubated with brief EdU can be used to study cell cycle dynamics.Note: The optimal incubation time is related to the cell cycle. Most tumor cell lines can use a 2-hour incubation time, as shown in Appendix 2. The concentration of EdU is related to the incubation time, and high concentrations such as 10-50 should be used for short-term incubation (<2 hours) µ M; Long term incubation (>24 hours) should use low concentrations, such as 1-10 µ M; You can also refer to Appendix 3.4. Cell fixation and permeation promotionNote: For experiments that require cell surface antigen labeling, it can be considered to wash cells twice with 1% BSA after completing EdU incubation, before cell fixation and permeation promotion.(1) After incubation, collect cells, add 1 mL of PBS to each tube to clean the cells, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant to remove EdU residue that has not been added to DNA.(2) Add 1 mL of 4% paraformaldehyde fixative to each tube to resuspend cells.(3) Incubate at room temperature for 20 minutes, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant.(4) Add 1 mL of 2 mg/mL glycine to each tube and incubate for 5 minutes. Neutralize the remaining fixed solution, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Add 1 mL of PBS to each tube for cleaning once, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant.(5) Add 1mL of 0.5% Triton X-100 osmotic enhancer to each tube and resuspend cells. Incubate at room temperature for 10 minutes.5. EdU detectionNote: For 6-well plate samples, reference can be made to 1 mL of working solution per well. Users can adjust the dosage according to their own sample situation.(1) Prepare 1 x Click iT EdU reaction buffer: Dilute component C 10 times with ddH2O.(2) Prepare 5 x Click iT EdU buffer additives (component E): Add 300 µ L ddH2O to 30 mg of component E in a test tube (final concentration 100 mg/mL), mix well until completely dissolved. After use, the remaining storage solution is stored at -20 ℃ and can be stored for one year. Once the solution turns brown, it indicates that the active ingredients have degraded and cannot be reused.Note: Different specifications of component E are dissolved in ddH2O according to this ratio to form 5 x storage solution for future use.(3) Prepare 1 x Click iT EdU buffer additive: Dilute 5 x Click iT EdU buffer additive storage solution with ddH2O to 1 x, and the solution should be prepared and used immediately.(4) Prepare Click it working solution according to Table 2.Table 2 Click it working fluidReaction componentsVolume of liquid required for a single reaction1×Click-iT EdU Reaction buffer875 µLCuSO4 (Component D)20 µLYF® 488/555/594/647A Azide(Component B)5 µL1×Click-iT EdU Buffer additives100 µLTotal volume1 mL(5) Soak at 1000 rpm for 5 minutes, discard the supernatant, remove the enhancer, add 1mL of 1% BSA washing solution to each tube and wash twice. Soak at 1000 rpm for 5 minutes, discard the supernatant.(6) Add 1 mL of Click iT working solution to each tube and mix well.(7) Incubate at room temperature in dark for 30 minutes.(8) Soak at 1000 rpm for 5 minutes, discard the staining reaction solution, add 1% BSA to each tube to wash the cells twice, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the cells again with 1 mL of 1% BSA (the volume of resuspend cells can be adjusted according to the number of cells), and detect with a flow cytometer.Note: If other biomarker tests are required, please refer to step 4.6. Intracellular antigen labeling (optional steps)(1) Add antibody working solution and mix well.(2) Under dark conditions, incubate antibodies at appropriate temperature and time.7. Flow detection and analysis:(1) It is recommended to conduct flow cytometry testing immediately after dyeing is completed; If conditions are limited, please store in a dark place at 4 ℃ for testing, but it should not exceed 3 days.(2) It is recommended to test the number of cells up to one million levels as much as possible. If the number of cells is small, the number of cells tested can be adjusted to 100000 levels starting from the experiment. For cases where the cell yield is too low (just to the level of ten thousand), it may not be conducive to making a flow chart. Therefore, the cleaning frequency in step 5 (8) can be appropriately reduced.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. fluorescent dyes have quenching problems. Please try to avoid light during experimental operation to slow down fluorescence quenching. 3. click it edu buffer additive solution should be prepared and used immediately to ensure the best results. 4. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Cell proliferation detection (cell imaging flow universal)... Read More | D665729 Component 50 T Storage D665729A Conversion Buffer CR 5×1 mL RT D665729B Buffer CL 30 mL RT D665729C Buffer MD 0.4 mL RT D665729D Buffer DB 10 mL RT D665729E Buffer WB (concentrate) 10 mL RT D665729F Buffer GW1 (concentrate) 13 mL RT D665729G Buffer GW2 (concentrate) 15 mL RT D665729H D665729 Component 50 T Storage D665729A Conversion Buffer CR 5×1 mL RT D665729B Buffer CL 30 mL RT D665729C Buffer MD 0.4 mL RT D665729D Buffer DB 10 mL RT D665729E Buffer WB (concentrate) 10 mL RT D665729F Buffer GW1 (concentrate) 13 mL RT D665729G Buffer GW2 (concentrate) 15 mL RT D665729H Buffer EB 4 mL RT D665729I Buffer PS 10 mL RT D665729J Spin Columns DF 50 Pcs 2-8 ℃ D665729K Collection Tubes 50 Pcs RTProduct Introduction:The basic principle of this reagent kit is that after DNA is treated with sodium bisulfite, unmethylated cytosine can be transformed into uracil, while methylated cytosine remains unchanged. And adopting an innovative high-temperature treatment method, the transformation time is greatly shortened, the transformation efficiency is improved, and the transformation efficiency can reach over 99%. At the same time, using a silicon-based membrane purification column, DNA can be recovered and purified from the methylated solution through a simple binding washing elution step. The recovered DNA has high purity and good integrity, and can be directly used for sequencing, methylated PCR detection, chip analysis, connection and transformation, enzyme digestion, labeling, microinjection, PCR and in vitro transcription and other molecular biology experiments.Self prepared reagents: anhydrous ethanol, 75% ethanol.Preparation and important precautions before the experiment1. Product usage method:(1) 10 times packaging preparation method: CT Conversion Agent is a solid mixture that must be prepared before first use. Add 2 ml sterile water and 100 µ M-Dissolving Buffer and 300 µ Add M-Diffusion Buffer to the CT Conversion Agent tube. Dissolve at 55 ° C and shake until completely dissolved. Store the CT Conversion Agent solution at room temperature (20 ° C-30 ° C) in the dark before use. The CT Conversion Agent for each tube is designed for 10 DNA treatments. In order to achieve better results, the prepared CT Conversion Agent should be used immediately. If not used immediately, the CT Conversion Agent solution can be stored at -20 ° C for 1 week. Before use, be sure to thaw the stored CT Conversion Agent solution at room temperature and mix thoroughly by shaking or inverting for 2 minutes, CT Conversion Reagent is sensitive to light, so it is important to minimize exposure to light as much as possible.(2) 50 times packaging preparation method: CT Conversion Agent and M-Dissolving Buffer are solid mixtures that must be prepared before first use. Add 5 ml of sterile water to the M-Dissolving Buffer and shake to dissolve. After all the solids have dissolved, transfer all the solution from the M-Dissolving Buffer tube to the CT Conversion Agent tube and add 5.5 ml of sterile water. Add 1.5 ml of M-Dilution Buffer to the CT Conversion Agent tube. Dissolve at 55 ° C and shake until completely dissolved. Store the CT Conversion Agent solution at room temperature (20 ° C-30 ° C) in the dark before use. The CT Conversion Agent for each tube is designed for 50 DNA treatments. In order to achieve better results, the CT Conversion Agent should be used immediately after preparation. If not immediately used, the CT Conversion Agent solution can be stored at -20 ° C for 1 week. Before use, be sure to thaw the stored CT Conversion Agent solution at room temperature and mix thoroughly by shaking or inverting for 2 minutes, CT Conversion Reagent is sensitive to light, so it is important to minimize exposure to light as much as possible.2. Before the first use, anhydrous ethanol should be added to the M-Wash Buffer according to the instructions on the reagent bottle label.Operation stepsThe range of DNA prepared each time is 1 ng-4 µ Between g, the optimal amount is 500 ng-2 µ G.1. Take 20 µ Add DNA sample into centrifuge tube (self provided), and if the sample amount is insufficient, replenish with water up to 20 µ L.2. Add 2.2 to the DNA sample µ Mix the sample well with the M-Dilution Buffer of l.3.42 ℃ water bath for 30 minutes.4. Add 220 to the sample obtained from the previous step µ Prepare the CT Conversion Agent solution, mix well, and incubate in an 80 ℃ constant temperature water bath in a dark place for 60 minutes.5. Add 480 to the solution in the previous step µ M - Buffer PA, gently mix upside down.6. Column balance: Add 200 to the spin columns DS that have been loaded into the collection tube µ Centrifuge at 12000 rpm (~13400 × g) for 2 minutes, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.7.Add all the solution obtained from step 5 to the adsorption column (already loaded into the collection tube), let it stand at room temperature for 2 minutes, 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.Attention: The maximum capacity of the adsorption column is 750 µ l. If the sample volume is greater than 750 µ L can be added in batches.8. Add 500 to the adsorption column µ Centrifuge at 12000 rpm for 1 minute using M-Buffer PA, discard the waste liquid from the collection tube, and place the adsorption column in the recovery tube.9. Add 650 to the adsorption column µ M-Wash Buffer (please check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column in the collection tube.10.12000 rpm for 2 minutes, discard the waste liquid, and 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 will affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).11. Place the adsorption column into a new centrifuge tube (provided by oneself), and add 20 drops to the middle position of the adsorption membrane in the air µ M-Elution Buffer (pH 8.5), leave at room temperature for 2 minutes. Collect DNA solution by centrifugation at 12000 rpm for 1 minute.12. Collect 20 µ Add 2.2 to DNA µ M-Diffusion Buffer, let it stand at room temperature for 30 minutes.13. Add 500 to the solution µ After pre cooling anhydrous ethanol, invert and mix well, and place the solution at -20 ℃ to precipitate for 30 minutes (overnight precipitation is more effective).14.12000 rpm for 15 minutes and gently discard the supernatant.15. Add 75% ethanol, centrifuge at 12000 rpm for 1 minute, pour out the supernatant, wait for ethanol to evaporate at room temperature, then add 20 µ Dissolve the M-Elution buffer and store the DNA at -20 ℃. The DNA collected in this step can be used for subsequent related experiments... Read More | Products contentN665968Component96 TStorageN665968Adex N501-N508 Primers for Illumina 8×12 µL-20℃. Avoid freeze/thaw cycle.N665968BIndex N701-N712 Primers for Illumina 12×8 µL-20℃. Avoid freeze/thaw cycle. Products IntroductionThis kit is a companion kit for the Products contentN665968Component96 TStorageN665968Adex N501-N508 Primers for Illumina 8×12 µL-20℃. Avoid freeze/thaw cycle.N665968BIndex N701-N712 Primers for Illumina 12×8 µL-20℃. Avoid freeze/thaw cycle. Products IntroductionThis kit is a companion kit for the transposase-based second-generation sequencing Rapid DNA Library Construction Kit, designed for Illumina platform library construction, which contains 8 primers at the N5 end and 12 primers at the N7 end, which can be used to prepare 96 different bipartite Index libraries. 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. The prepared libraries can be sequenced 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 and 1.5 ml centrifuge tubes; tips: It is recommended to use high-quality filtration tips to prevent contamination of reagent 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.For 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-N508 Primers for IlluminaIndex N701-N712 Primers for Illumina... Read More | This kit is used to extract and purify high-quality total RNA from various plants, and is also suitable for the extraction of fungal hyphal RNA. A unique Shredder separation column is used for homogenization and filtration of high viscosity plant or fungal lysates, while silica based membrane is This kit is used to extract and purify high-quality total RNA from various plants, and is also suitable for the extraction of fungal hyphal RNA. A unique Shredder separation column is used for homogenization and filtration of high viscosity plant or fungal lysates, while silica based membrane is used to adsorb RNA for purification, effectively removing various pollutants such as polysaccharides through washing. The washed RNA can be directly used in various downstream experiments. RNA with a molecular weight greater than 200 bases was extracted using this reagent kit, with high purity and almost no DNA residue. If it is an RNA experiment that is very sensitive to trace amounts of DNA, the remaining DNA can be digested and removed on a column using DNase I without RNase. The extracted RNA can be used for experiments such as Northern Blot, Dot Blot, RT-PCR, and in vitro translation. R665489Component50 TStorageR665489ABuffer RL35 mLRTR665489BBuffer RLC35 mLRTR665489CBuffer RW140 mLRTR665489DBuffer RW2 (concentrate)11 mLRTR665489ERNase-Free Water10 mLRTR665489FSpin Columns FL with Collection Tubes50 setsRTR665489GSpin Columns RM with Collection Tubes50 setsRTR665489HRNase-Free Centrifuge Tubes (1.5 mL)50 EART Self prepared reagents:β- Mercaptoethanol, anhydrous ethanol (newly opened or dedicated for RNA extraction).Preparation and important precautions before the experiment: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.2. The extracted samples should avoid repeated freeze-thaw cycles, otherwise it will affect the quantity and quality of RNA extraction.3. Please add Buffer RL before use β- Mercaptoethanol, with a final concentration of 1%. Add 10 to 1 ml Buffer RL µ L β Mercaptoethanol. join β- The buffer RL room temperature of mercaptoethanol can be stored for one month. No need to add buffer RLC when using it β- Mercaptoethanol.Before the first use, anhydrous ethanol should be added to Buffer RW2 according to the instructions on the reagent bottle label.5. If precipitation occurs in Buffer RL and Buffer RLC, please heat them to dissolve and place them 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.7. If downstream experiments are highly sensitive to DNA, it is recommended to treat RNA with DNase I without RNase.Operation steps:1. Take 50-100 mg of fresh plant tissue, add liquid nitrogen and quickly grind it into powder.2. Collect the ground powder into a centrifuge tube (provided by oneself) and add 600 µ L Buffer RL (check if it is added before use) β- Sulfhydryl ethanol or Buffer RLC, vortex oscillation causes it to fully decompose.Attention:1) The main component of Buffer RL is guanidine isothiocyanate, which is suitable for the lysis of most plant tissues. However, in some plant tissues (such as corn endosperm), due to the unique secondary metabolites, guanidine isothiocyanate causes precipitation in the sample, resulting in poor RNA extraction efficiency. In this case, Buffer RLC can be added instead of Buffer RL.2) Incubating at 56 ℃ for 1-3 minutes helps with tissue lysis, but plants with high starch content should not be subjected to high-temperature incubation.3. Transfer all the liquid obtained in step 2 to the spin columns FL that have been loaded into the collection tube, centrifuge at 12000 rpm (~13400 × g) for 2 minutes, and transfer the supernatant from the collection tube to a new centrifuge tube (provided by oneself).Attention:1) When aspirating liquid, the tip of the gun can be cut off for easy sampling.2) Spin Columns FL can remove most of the fragments, but there will still be a small amount flowing out. After centrifugation, precipitation will form in the collection tube. When proceeding to the next step, be careful not to absorb the sediment.4. Add 0.5 times the volume of anhydrous ethanol to the clean cracking solution obtained in step 3 and quickly mix well. Attention: Adding ethanol may cause precipitation, but it does not affect subsequent experiments.5. Add all the solutions obtained in step 4 to the spin columns RM that have been loaded into the collection tube. If it is not possible to add all the solutions to the adsorption column at once, please transfer them in two separate steps. Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.6. Add 700 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. Optional steps: If conducting RNA experiments that are highly sensitive to trace amounts of DNA, replace step 6 with the following steps.1) Add 350 to the adsorption column µ L Buffer RW1, centrifuge at 12000 rpm for 15 seconds, discard the waste liquid, and place the adsorption column back into the recovery manifold.2) Preparation of DNase I mixture: Take 52 µ Add 8 RNase Free Water to it µ 10 x Reaction Buffer and 20 µ DNase I (1 U/ µ l) Mix well and prepare to a final volume of 80 µ The reaction solution of L.Attention:The above system is configured according to our company's DNase I reaction system. Please refer to the corresponding instructions for other company products.3) Add 80 µ l of DNase I reaction solution directly to the adsorption column and incubate at 20-30 ℃ for 15 minutes.4) Add 350 to the adsorption column µ L Buffer RW1, centrifuge at 12000 rpm for 15 seconds, discard the waste liquid, and place the adsorption column back into the recovery manifold.7. Add 500 to the adsorption column µ Buffer RW2 (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 15 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.8. Repeat step 7.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 dry the anhydrous ethanol in the column.Attention: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.).10. Place the adsorption column in a new RNase free centrifuge tube, and add 30-50 to the middle of the adsorption column in the air µ Place RNase Free Water at room temperature for 1 minute, 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 30 µ l. Small volume affects the recovery rate.2) If you want to increase RNA production, you can use 30-50 µ Repeat step 10 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 10... Read More |