| Description | Animal liver and kidneys are the main organs for amino acid metabolism. Therefore, changes in urinary amino acids best reflect the physiological state of the liver and kidneys. Additionally, amino acid levels can indicate conditions such as burns and typhoid fever. The amino acid content in plants Animal liver and kidneys are the main organs for amino acid metabolism. Therefore, changes in urinary amino acids best reflect the physiological state of the liver and kidneys. Additionally, amino acid levels can indicate conditions such as burns and typhoid fever. The amino acid content in plants is significant for studying nitrogen metabolism changes under different conditions and during various growth and development stages, as well as for understanding nitrogen absorption, transport, assimilation, and nutritional status in plants.Detection Principle: The α-amino group of amino acids reacts with ninhydrin hydrate to produce a blue-purple compound with a characteristic absorption peak at 570 nm. The amino acid content is calculated by measuring the absorbance at 570 nm.Detection Range: 0.625 - 40 µmol/mLSensitivity: 0.5 µmol/mLApplicable Samples: Serum (plasma), animal/plant tissues, cells, cell culture supernatants, bacteria, urineG1501758Component96TStorageG1501758AExtraction Buffer100 mL2-8℃G1501758BAssay Buffer10 mL2-8℃G1501758CSubstrate1EA2-8℃. Store in the dark.G1501758DStandard (10mg Cysteine)1EA2-8℃. Store in the dark.User-Prepared Instruments and ReagentsMicroplate reader or visible spectrophotometer (capable of measuring absorbance at 570 nm)96-well plate or micro glass cuvettes, adjustable micropipettes and tipsRefrigerated centrifuge, water bathDeionized water, EthanolHomogenizer (for tissue samples)Experimental Procedure1. Reagent PreparationReagent NameReagent PreparationNotesExtraction BufferReady-to-use; Equilibrate to room temperature before use.Store at 4°C.Assay BufferReady-to-use; Equilibrate to room temperature before use.Store at 4°C. Toxic and irritant. Perform experiments in a fume hood.SubstrateToxic and irritant. Perform experiments in a fume hood.Working SubstratePrepare before use: Dissolve in 4 mL of 95% Ethanol.Unused dissolved substrate can be stored at 4°C protected from light for one week. For long-term storage, aliquot and store at -20°C protected from light for one month. Avoid repeated freeze-thaw cycles.StandardPrepare before use: Add 2.066 mL deionized water to dissolve completely, resulting in a 40 µmol/mL stock.Unused dissolved standard can be stored at 4°C for one week. For long-term storage, aliquot and store at -20°C for one month. Avoid repeated freeze-thaw cycles.2. Standard Curve SetupDilute the 40 µmol/mL standard stock solution with deionized water to concentrations of 20, 10, 5, 2.5, 1.25, and 0.625 µmol/mL as shown in the table below.TubeStandard VolumeDeionized Water Volume (µL)Standard Concentration (µmol/mL)Std.1200µL of 40µmol/mL040Std.2100µL of Std.110020Std.3100µL of Std.210010Std.4100µL of Std.31005Std.5100µL of Std.41002.5Std.6100µL of Std.51001.25Std.7100µL of Std.61000.625Note: The standard curve must be generated with each experiment. Diluted standard solutions are unstable and must be used within 4 hours.3. Sample PreparationNote: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to one month.3.1 Animal TissueWeigh approximately 0.1 g of tissue. Add 1 mL of Extraction Buffer and homogenize thoroughly at room temperature. Transfer the homogenate to a 1.5 mL microcentrifuge tube. Cap tightly (to prevent moisture loss) and incubate in a boiling water bath for 15 minutes. Cool with tap water. Centrifuge at 10,000 rpm for 10 minutes at room temperature. Collect the supernatant for assay.3.2 Plant TissueWeigh approximately 0.1 g of tissue. Add 1 mL of Extraction Buffer and grind. Sonicate for 5 minutes at room temperature (power 20% or 200W, pulse 3s on, 7s off, repeat 30 times). Transfer to a 1.5 mL microcentrifuge tube. Cap tightly (to prevent moisture loss) and incubate in a boiling water bath for 15 minutes. Cool with tap water. Centrifuge at 10,000 rpm for 10 minutes at room temperature. Collect the supernatant for assay.3.3 Cells or BacteriaCollect 5 million cells or bacteria into a centrifuge tube. Wash cells with cold PBS, centrifuge, and discard the supernatant. Add 1 mL of Extraction Buffer. Sonicate for 5 minutes at room temperature (power 20% or 200W, pulse 3s on, 7s off, repeat 30 times). Transfer to a 1.5 mL microcentrifuge tube. Cap tightly (to prevent moisture loss) and incubate in a boiling water bath for 15 minutes. Cool with tap water. Centrifuge at 10,000 rpm for 10 minutes at room temperature. Collect the supernatant for assay.3.4 Serum (Plasma), Cell Culture Supernatant, Urine, and Other LiquidsPipette 0.5 mL of the liquid sample and add 0.5 mL of Extraction Buffer. Cap tightly (to prevent moisture loss) and incubate in a boiling water bath for 15 minutes. Cool with tap water. Centrifuge at 10,000 rpm for 10 minutes at room temperature. Collect the supernatant for assay.Note: If protein concentration measurement is required, use Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648).4. Assay Steps4.1 Preheat the microplate reader or visible spectrophotometer for at least 30 minutes. Set the wavelength to 570 nm. For spectrophotometers, zero the instrument with deionized water.4.2 Sample Measurement (Add reagents sequentially into microcentrifuge tubes as below):ReagentBlank Tube (µL)Standard Tube (µL)Test Tube (µL)Deionized Water1000Standard (various conc.)0100Sample0010Working Substrate202020Assay Buffer5050504.3 Mix well and cap the tubes tightly (to prevent moisture loss). Incubate in a boiling water bath for 5 minutes. Cool in tap water for 10 seconds. Add 120 µL of 60% ethanol to each tube and invert several times to mix. Transfer 150 µL from each tube to the corresponding wells of a 96-well plate or micro glass cuvette. Measure the absorbance at 570 nm, recorded as A blank, A standard, and A test. Calculate ΔA test = A test - A blank and ΔA standard = A standard - A blank (The blank tube only needs to be prepared once). All measurements must be completed within 30 minutes after color development. Note:It is recommended to perform a preliminary test with 2-3 samples expected to have significant differences before the formal experiment. If A <sub> test </sub> is greater than 2.0, dilute the sample further with deionized water and multiply the result by the dilution factor.Proline and hydroxyproline do not produce an absorption peak at 570 nm when reacting with ninhydrin. Therefore, the results measured at 570 nm do not include these two amino acids.5. Calculation of ResultsNote: We provide two formulas, including the derived formula and a simplified version. They are equivalent. It is recommended to use the simplified formula in bold for final calculation.5.1 Standard Curve PlottingPlot the standard concentration (y-axis) against ΔA standard (x-axis) to generate the standard curve. Substitute ΔA test into the standard curve equation to calculate y (µmol/mL).5.2 Sample Amino Acid Content Calculation(1) Based on Sample WeightAmino Acid Content (µmol/g weight) = y ÷ (W ÷ V<sub>extraction</sub>) × n = y ÷ W × n(2) Based on Protein ConcentrationAmino Acid Content (µmol/mg prot) = y ÷ Cpr × n(3) Based on Bacterial or Cell CountAmino Acid Content (µmol/10⁴ cells) = y ÷ (Count ÷ V<sub>extraction</sub>) × n = y ÷ 500 × n = 0.002 × y × n(4) Based on Liquid VolumeAmino Acid Content (µmol/mL) = y × 2 × nParameter Definitions:W: Sample weight (g)V extraction : Volume of Extraction Buffer added (1 mL)n: Sample dilution factorCpr: Protein concentration of the supernatant (mg/mL)500: Total number of bacteria or cells (5 million)2: Dilution factor for liquid samples [(0.5 mL sample + 0.5 mL Buffer) / 0.5 mL sample = 2]6. Representative ResultsTypical Standard Curve: y = 20.349x - 0.423, R² = 0.9971 Figure 1: Total Amino Acid Standard Curve Precautions1. Biochemical reagents are generally irritating, biologically toxic, etc. For your safety and health, please use appropriate biosafety precautions throughout the experiment, including wearing lab coats, masks, gloves, head covers, etc. Perform experiments in a fume hood or biosafety cabinet.2. This product is for research use only. Not for use in clinical diagnosis... Read More | Product introduction：Product introduction：Cell Cycle Assay Kit Plus ( Cell Cycle Assay Kit Plus ) has certain applicability for live cells and fixed cell cycle detection. For different types of cells, whether it is applicable or not needs to be determined after testing. Cell Cycle Product introduction：Product introduction：Cell Cycle Assay Kit Plus ( Cell Cycle Assay Kit Plus ) has certain applicability for live cells and fixed cell cycle detection. For different types of cells, whether it is applicable or not needs to be determined after testing. Cell Cycle Assay Kit Plus ( Cell Cycle Assay Kit Plus ) uses RedNucleus I staining to detect cell cycle. RedNucleus I is a far-infrared nucleic acid dye with cell membrane permeability, which can quickly enter living cells, specifically bind to DNA, and perform cell cycle detection on living cells without RNase digestion. Compared with the traditional PI staining method, the cells do not need to be broken or fixed, and the operation is simpler. RedNucleus I is a fluorescent dye of double-stranded DNA, and the fluorescence intensity after binding to double-stranded DNA is proportional to the content of double-stranded DNA. The intracellular DNA content can be measured by flow cytometry, and then the cell cycle analysis can be carried out according to the distribution of DNA content. After RedNucleus I staining, assuming that the fluorescence intensity of G0 / G1 phase cells is 1, the theoretical value of the fluorescence intensity of G2 / M phase cells containing two copies of genomic DNA is 2, and the fluorescence intensity of S phase cells undergoing DNA replication is between 1-2. In addition, RedNucleus I is compatible with dyes such as Horizon BV / BUV, FITC and R-PE, and can be periodically detected after sample staining.The kit is usually used to detect the cell cycle of cultured adherent or suspended cells. If it is used for cell cycle detection of tissues, the tissues must be digested into a single cell state.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. this product is applicable to the detection of living cells and fixed cell cycle with certain limitations. Whether it is applicable to different types of cells needs to be determined after testing. If fixation is needed, it is recommended to use ice bath pre cooling 75-80% ethanol -20 ℃ to fix cells overnight. 3. fluorescent dyes have quenching problems. Please try to avoid light during storage and use to slow down fluorescence quenching. 4. for your safety and health, please wear experimental clothes and disposable gloves.Instruction： Experimental materials ( self-provided )：①cell lines or other cell samples ( self-prepared ) ；②This kit ; ③ trypsin ( self-prepared ) ;④ Cell culture medium containing FBS ( self-prepared ) ; Experimental procedure： 1.Preparation of cell samples : ( 1 ) ( This step is for adherent cells, if suspended cells, can be carried out directly step ( 2 ) ) Digest cells with trypsin, add cell culture medium, gently blow away cells, collected into the centrifuge tube. Note : The number of cells on the machine needs to reach 50,000 and above, so the initial number of cells collected needs to be sufficient. ( 2 ) Centrifuged about 1000 g for 3-5 min to precipitate cells. Carefully remove the supernatant, add about 1 mL of ice bath pre-cooled 1 × staining buffer ( 10 × staining buffer diluted with diH2O at 1 : 10 ), re-suspend the cells. Repeat once. ( 3 ) Centrifuged about 1000 g for 3-5 min to precipitate cells. After the supernatant was discarded, 1 mL of culture medium was added to re-suspend the cells ( for fixed cells, 1 × PBS can also be used to re-suspend ). Gently flick the bottom of the centrifuge tube to properly disperse the cells to avoid cell aggregation. 2.Staining : 4 µL of RedNucleus I staining solution was added to each tube of cell samples, slowly and fully mixed, and incubated at room temperature in dark for 20 min ( or incubated at 37 ° C in dark for 5-10 min ). The optimal incubation time of different cells is different, and the staining time can be adjusted and optimized according to the actual staining effect to obtain a more ideal staining effect. 3.Flow cytometry detection and analysis : Excited at 638 nm by flow cytometry, it is recommended to detect in RL3 or FL4 channels, or use RL1 and RL2 channels. Cell DNA content analysis and light scattering analysis were performed using appropriate analysis software.Scope of application：Cell cycle detection... Read More | 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 | Products Content:F666101Component500 U5000 UStorageF666101AFastStar Probe Buffer (for bisDNA)2×1.2 mL2×12 mL-20℃. Avoid freeze/thaw cycle. Protect from light.F666101BSuperFastStar DNA Polymerase (5U/µL)100 µL1 mL-20℃. Avoid freeze/thaw cycle. Protect from light.Products Content:F666101Component500 U5000 UStorageF666101AFastStar Probe Buffer (for bisDNA)2×1.2 mL2×12 mL-20℃. Avoid freeze/thaw cycle. Protect from light.F666101BSuperFastStar DNA Polymerase (5U/µL)100 µL1 mL-20℃. Avoid freeze/thaw cycle. Protect from light.Products IntroductionThis product is mainly used for PCR using bisulfite-treated DNA as template, in which SuperFastStar DNA Polymerase is a new high-efficiency hot-start enzyme modified by bis-monoclonal antibody, which is completely blocked at room temperature, thus effectively avoiding non-specific amplification caused by the non-specific binding of the primer to the template or the primer dimerization under the condition of room temperature. The optimized FastStar Probe Buffer (for bisDNA) contains PCR Buffer, dNTPs and Mg2+, etc., which is easy to use as customers only need to add templates, primers and probes.caveat1 Before use, please mix the product gently by turning it up and down after it has been completely melted and centrifuged briefly.2. Avoid repeated freezing and thawing of the product, which may degrade its performance. This product can be stored at -20℃ for a long period of time, protected from light. If frequent use is required within a short period of time, it can be stored at 2-8℃.Usage The following examples are conventional PCR reaction systems and conditions, which should be improved and optimized according to the template, primer structure and target fragment size.1.PCR reaction system Note: 1) Usually, better results can be obtained with a primer concentration of 0.2 µM, and 0.1-1.0 µM can be used as a reference for setting the range.2）The concentration of the probe used is related to the fluorescence quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, so please refer to the instrument manual or the specific requirements for the use of each fluorescent probe to adjust the concentration.3）Usually the amount of DNA template is 10-100 ng of genomic DNA or 1-10 ng of cDNA as a reference. Since the templates of different species contain different copy numbers of the target gene, the templates can be diluted in gradients to determine the optimal amount of template to use.2.PCR reaction conditionsNote: 1) The initial denaturation of this product at 95°C for 30s is sufficient for enzyme activation; complex templates can be extended to 3min denaturation.(2) It is recommended to use two-step PCR reaction program, if you can't get good experimental results due to the use of primers with lower Tm value, etc., you can try three-step PCR amplification, and the annealing temperature should be set in the range of 56℃-64℃ as a reference... 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 |