| Description | Glutathione is a tripeptide containing a γ-amide bond and a sulfhydryl group, composed of glutamate, cysteine, and glycine. It is widely found in animal and plant tissues and microorganisms. In living organisms, it helps maintain normal immune system function and has antioxidant and Glutathione is a tripeptide containing a γ-amide bond and a sulfhydryl group, composed of glutamate, cysteine, and glycine. It is widely found in animal and plant tissues and microorganisms. In living organisms, it helps maintain normal immune system function and has antioxidant and detoxifying effects. Glutathione exists in two forms: reduced (GSH) and oxidized (GSSG). GSSG, also known as glutathione disulfide, is formed by the oxidation of two glutathione molecules. GSSG can be reduced back to GSH by glutathione reductase; therefore, it exists primarily in the reduced form in organisms. The ratio of reduced to oxidized glutathione (GSH/GSSG) serves as a key dynamic indicator for assessing the cellular redox state. Detection Principle: Endogenous GSH in the sample is masked by 2-vinylpyridine. Under the catalysis of glutathione reductase (GR), GSSG is reduced to GSH. The generated GSH then reacts with 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB) to produce yellow-colored 5-thio-2-nitrobenzoic acid (TNB), which has a characteristic absorption peak at 412 nm. The GSSG content is quantified by measuring the change in absorbance. Detection Range: 1-20 µM Sensitivity: 1 µM Applicable Samples: Animal/plant tissues, blood cells, cells, bacteria, serum (plasma).O1492795Component96TStorageO1492795AExtraction Buffer70 mL×22-8℃O1492795BInhibitor210 µL-20℃. Store in the dark.O1492795CAssay Buffer20 mL2-8℃O1492795DGR14 µL2-8℃. Store in the dark.O1492795EGR Cofactor2 EA-20℃. Store in the dark.O1492795FChromogen2 EA2-8℃. Store in the dark.O1492795GStandard1 EA2-8℃. Store in the dark.User-Provided Instruments and ReagentsTypeNameNotesInstrumentMicroplate ReaderCapable of measuring absorbance at 412 nm.Consumables96-well MicroplateStandard transparent plate.ReagentsPBS / Deionized WaterFor washing samples / Preparing reagents.OthersHomogenizer (for tissue samples), water bath, ice bucket, low-temperature centrifuge, adjustable pipettes and tipsUsing a multichannel pipette for large-scale detection can improve efficiency.Experimental Procedure1. Reagent PreparationReagent NameReagent PreparationPrecautionsExtraction BufferReady-to-use; equilibrate to room temperature before use.Store at 4°C.Diluted Extraction BufferAdd 500 µL Extraction Buffer to 4.5 mL deionized water.Obtained by 10-fold dilution of Extraction Buffer.InhibitorReady-to-use; equilibrate to room temperature before use.Store at -20°C protected from light. Toxic and irritant; recommended to handle in a fume hood.Assay BufferReady-to-use; equilibrate to room temperature before use.Store at 4°C.GR DilutionBefore use, prepare by adding 1 µL GR to 20 µL deionized water per sample.Prepare freshly before use.GR Cofactor DilutionBefore use, add 1.5 mL deionized water to each vial; equilibrate to room temperature protected from light.After dissolution, store at -20°C protected from light for up to 1 month.Chromogen DilutionBefore use, add 1.5 mL deionized water to each vial; equilibrate to room temperature protected from light.After dissolution, store at 4°C protected from light for up to 1 month.GSSG StandardDissolve in 1 mL of Diluted Extraction Buffer.20 mM; After dissolution, aliquot and store at -20°C protected from light for up to 1 month.2. Standard PreparationTake 100 µL of the 20 mM GSSG standard and dilute with 900 µL Diluted Extraction Buffer to obtain a 2 mM GSSG standard solution.Take 10 µL of the 2 mM GSSG standard and dilute with 990 µL Diluted Extraction Buffer to obtain a 20 µM GSSG standard solution.Further dilute the standard as shown in the table below. A standard curve must be prepared for each experiment. Diluted standard solutions are unstable and must be used within 4 hours.Standard Working Solution20µM Standard (µL)Diluted Extraction Buffer (µL)Concentration (µM)110002028020163604012440608520804610902759513. Sample PreparationNote: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 10 days. Because the Extraction Buffer contains a protein precipitant, the supernatant cannot be used for protein concentration determination. If protein content needs to be measured, prepare another identical sample using deionized water instead of Extraction Buffer.3.1 Animal/Plant Tissue Samples:Use fresh tissue samples whenever possible. Weigh 0.1 g of tissue, add 1 mL of pre-cooled Extraction Buffer, and homogenize quickly on ice (pre-cool the homogenizer on ice). Centrifuge the homogenate at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection.3.2 Serum/Plasma Samples:Use fresh serum (plasma) whenever possible. Centrifuge the collected serum (plasma) at 600 g, 4°C for 10 min. Within 30 minutes, aspirate the supernatant into another tube. Add an equal volume of Extraction Buffer, mix, then centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection.3.3 Cell or Bacterial Samples:Use fresh cells (bacteria) whenever possible; avoid using frozen cells (bacteria). Collect 5×10⁶ cells (bacteria). Wash twice with 1 mL of pre-cooled PBS (resuspend in PBS, centrifuge at 600 g, 4°C for 10 min). Add 3 times the volume of Extraction Buffer relative to the cell (bacterial) pellet to resuspend the cells (bacteria). Disrupt by ultrasound on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection.Note: Cells can also be extracted using a freeze-thaw method (not suitable for bacteria): Resuspend cells and subject to 2-3 rapid freeze-thaw cycles (freeze in liquid nitrogen, thaw in a 37°C water bath). Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection.4. Assay Steps4.1 Microplate Reader Preparation: Preheat for at least 30 minutes, set wavelength to 412 nm.4.2 Assay System Setup (Step 1 - Pre-treatment): Perform the following operations in 1.5 mL EP tubes. This step must be done in EP tubes. Do not add Inhibitor directly to the 96-well plate as it may corrode the plate. Inhibitor is toxic and irritant; recommended to handle in a fume hood.ReagentBlank Tube (µL)Standard Tube (µL)Test Tube (µL)Sample003Deionized Water30027Standard0300Inhibitor1.51.51.54.3 Mix well and incubate at 37°C for 30 minutes. This becomes the "Mixture".4.4 Assay System Setup (Step 2 - Reaction): Perform the following operations in a 96-well plate.ReagentBlank Well (µL)Standard Well (µL)Test Well (µL)Mixture212121Assay Buffer140140140GR Dilution222GR Cofactor Dilution202020Chromogen Dilution2020204.5 Absorbance Measurement: Mix thoroughly after addition. Read the absorbance at 412 nm (A1), recorded as A1 blank, A1 standard, and A1 test. Then incubate at 37°C protected from light for 10 minutes. Quickly read the absorbance at 412 nm again (A2), recorded as A2 blank, A2 standard, and A2 test. 5. Result Calculation The following provides both the derived formula and the simplified calculation formula, which are completely equivalent. 5.1 Data Processing Calculate ΔA = A2 - A1 for each. Then calculate ΔΔA standard = ΔA standard - ΔA blank And ΔΔA test = ΔA test - ΔA blank 5.2 Standard Curve Plotting 5.2 Standard Curve Plotting Plot the standard curve with standard concentration as the y-axis and ΔΔA standard as the x-axis. Substitute ΔΔA test into the equation to obtain the y value (µM). 5.3 Sample GSSG Content Calculation (1) Based on sample mass: GSSG (nmol/g) = y × V standard ÷ V sample × V extract ÷ W × n = 10 × y ÷ W × n (2) Based on cell or bacterial count: GSSG (nmol/10⁴) = y × V standard ÷ V sample × V extract ÷ 500 × n = 0.02 × y × V extract × n (3) Based on liquid volume: GSSG (nmol/mL) = y × V standard ÷ V sample × 2 × n = 20 × y × n (4) Based on protein concentration: GSSG (nmol/mg prot) = y × V standard ÷ V sample ÷ Cpr × n = 10 × y ÷ Cpr × n Parameter Description: 1 µM = 1 nmol/mL; V standard : Volume of standard added, 30 µL; V sample : Volume of sample added, 3 µL; V extract : Volume of Extraction Buffer added, 1 mL (for cells/bacteria, use the actual volume used); W: Sample mass, g; n: Sample dilution factor; Cpr: Sample protein concentration, mg/mL; 500: Cell or bacterial count, in units of 10⁴; 2: Dilution factor for liquid samples (added equal volume of Extraction Buffer).6. Result PresentationTypical Standard Curve: y = 8.0042x + 0.212, R² = 0.9997Example-1: 0.1 g of rat liver tissue was processed and assayed according to the procedure using a 96-well plate. Measured: ΔA test = A2 test - A1 test = 0.386 - 0.120 = 0.266 ΔA blank = A2 blank - A1 blank = 0.132 - 0.097 = 0.035 ΔΔA test = ΔA test - ΔA blank = 0.266 - 0.035 = 0.231 Substituting ΔΔA test into the standard curve equation gives y = 2.061 µM. Calculated based on sample mass: GSSG (nmol/g) = y × V standard ÷ V sample × V extract ÷ W × n = 10 × y ÷ W × n = 206.1 nmol/g.Precautions1. It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.2. The samples extracted with this kit are suitable for the detection of oxidized glutathione (GSSG). Because the extraction buffer contains a protein precipitant, the supernatant cannot be used for protein concentration determination. If protein content needs to be measured, prepare another identical sample using deionized water instead of the extraction buffer. For protein concentration determination, Aladdin BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) are recommended.3. This kit is compatible with spectrophotometer detection. Adjust the preparation volume of detection reagents proportionally according to the spectrophotometer's requirements.4. It is recommended to establish your own standard curve for improved accuracy. If not, you may refer to the typical standard curve formula provided in the results section for calculation.5. Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please wear appropriate personal protective equipment (lab coat, mask, gloves, hair cap, etc.) throughout the experiment and perform experiments in a fume hood or biosafety cabinet.6. This product is for scientific research use only. Not intended for clinical diagnosis.Frequently Asked Questions Q: What should I do if the sample ΔA test is too high or too low? Frequently Asked Questions Q: What should I do if the sample ΔA test is too high or too low? A: If the sample ΔA test is greater than the ΔA standard of the 20 µM standard, the GSSG content in the sample is too high. Dilute the sample appropriately with deionized water (multiply by the dilution factor in the calculation). If the sample ΔA test is less than 0.005, increase the sample amount.Q: Can blood cell samples be detected?A: Yes, blood cell samples can be detected. Centrifuge the collected anticoagulated blood at 600 g, 4°C for 10 min. Discard the upper plasma and wash the pellet 2-3 times with 3 volumes of PBS (resuspend blood cells in PBS, centrifuge at 600 g, 4°C for 10 min). Add an equal volume of Extraction Buffer, mix, and let stand at 4°C for 10 min. Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection... Read More | Product content： O665490Component50 TStorageO665490ABlocking Buffer500 mL2-8℃. Do not freeze.O665490BAntibody Pretreat Solution (HRP/Rabbit)5×1 mL2-8℃. Do not freeze.O665490CDilution Buffer500 mL2-8℃. Do not freeze.O665490DWash Buffer (10×)500 mL2-8℃. Do not Product content： O665490Component50 TStorageO665490ABlocking Buffer500 mL2-8℃. Do not freeze.O665490BAntibody Pretreat Solution (HRP/Rabbit)5×1 mL2-8℃. Do not freeze.O665490CDilution Buffer500 mL2-8℃. Do not freeze.O665490DWash Buffer (10×)500 mL2-8℃. Do not freeze. Product Introduction：The one-step rapid WB assay kit (rabbit) is the latest Western Blot detection kit developed by Kangwei Century, which canObtain high-quality Western Blot results within about 1 hour, with simple operation, high detection sensitivity, low background, and noAdditional secondary antibodies need to be added, with strong system stability. The conventional Western Blot indirect detection process (blocking, primary antibody binding)Combining with secondary antibodies requires a long time, a complex experimental process, and requires multi-step optimization of conditions. The protein on the glue is transferred toAfter coating the carrier membrane, incubate it with the blocking solution in the reagent kit for 5 minutes, and then incubate the carrier with the primary antibody treated with antibody reaction solutionAfter washing the membrane three times (5 minutes each time), it can undergo luminescence or color detection. This reagent kit is designed for target protein oneThe use of an experimental system derived from rabbits.Notes:1. Customers need to prepare their own rabbit source primary antibody.2. Before using Blocking Buffer blocking solution, Antibody Pretreat Solution (HRP/Rabbit) antibody reaction solution (rabbit), 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 (rabbit) needs to be determined through preliminary experiments.6. Antibody reaction solution HRP (rabbit), 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. Antibodies with low specificity and affinity are not recommended for repeated use. 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/Rabbit) 100 µ Add rabbit 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 (rabbit) 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/Rabbit), and Dilution Buffer to the membrane (ensuring that the incubation solution completely submerges the membrane surface). Incubate at room temperature on a shaker at a speed of about 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.Example 1: Antigen 293T cell lysateA: Ordinary WB control: beta actin rabbit antibody (CW0097) 3.3ug incubated at room temperature for 40 minutes, washed with membrane, secondary antibody sheep anti rabbit HRP (CW0103) diluted at 1:10000, room temperature for 40 minutes, ECL (CW0049) exposed Example 2 Antigen is 293T cell lysateC: Ordinary WB control: PAK1, Epitomics rabbit monoclonal antibody 1:1000, incubated at room temperature for 40 minutes, washed with membrane, secondary antibody sheep anti rabbit HRP (CW0103) diluted at 1:10000, room temperature for 40 minutes, ECL (CW0049) exposedD: One step WB: Epitomics rabbit monoclonal antibody was incubated at 1:1000 room temperature for 40 minutes, and ECL (CW0049) was exposed... Read More | Product content Q665687Component100 TStorageQ665687AQuick T4 DNA Ligase (15 U/µL)100 µL-20℃. Avoid freeze/thaw cycle.Q665687B2×Quick Ligation Reaction Buffer5×200 µL-20℃. Avoid freeze/thaw cycle. Product IntroductionThe Quick Ligation Reaction Kit allows ligationProduct content Q665687Component100 TStorageQ665687AQuick T4 DNA Ligase (15 U/µL)100 µL-20℃. Avoid freeze/thaw cycle.Q665687B2×Quick Ligation Reaction Buffer5×200 µL-20℃. Avoid freeze/thaw cycle. Product IntroductionThe Quick Ligation Reaction Kit allows ligation of DNA sticky or flush ends in 5 minutes at room temperature (25°C). The kit contains Quick T4 DNA Ligase and 2×Quick Ligation Reaction Buffer optimized for fast and efficient DNA ligation.The ligation efficiency of Quick Ligation is equivalent to 1 hour of conventional ligation with T4 DNA Ligase. The Quick Ligation products can be used directly in routine bacterial transformation experiments.matters needing attention1. This kit enables most of the linkage reactions to reach the reaction endpoint within 5 minutes or less at 25°C. Increasing the reaction time will not enhance the reaction efficiency. If you use the rapid connection reaction after 1 hour, the conversion efficiency will be significantly reduced; if the rapid connection reaction at 25 ℃ overnight, the conversion efficiency will drop to 75%.2. 2×Quick Ligation Reaction Buffer contains ATP, which should be thawed on ice and mixed thoroughly before use. It is recommended to freeze the buffer in small tubes for the first time, so as to avoid repeated freezing and thawing, which will affect the efficiency of DNA ligation.3. Since T4 DNA Ligase contains glycerol, which is sticky and easy to hang on the wall, it is recommended to collect the liquid to the bottom of the tube by centrifugation for a short period of time before use, and the tip of the lance should not go too deep into the liquid surface when taking samples to avoid sticking to the tip of the lance and causing losses.4. If the quick ligation product is used for electrotransformation, the PEG in the quick ligation reaction system will affect the efficiency of electrotransformation, and it is recommended to use a centrifugal column to purify the ligation product from DNA before electrotransformation.Usage1. The reaction solution was prepared according to the following system:*The amount of Insert DNA used: the molar ratio of Vector DNA and Insert DNA is generally 1:3-1:8, and the appropriate molar ratio of Vector DNA and Insert DNA can be selected according to the experimental situation.Calculation of DNA molar number: DNA molar number (nmol)=DNA mass (ng)/( 660daltons x number of inserted DNA bases bp).2. mix gently and centrifuge briefly. react at 25°C for 5 minutes.Note: The reaction time should not exceed 15 minutes, otherwise the connection efficiency will be reduced.3. Do not perform heat inactivation reactions. Centrifuge instantly and collect the solution from the wall to the bottom of the tube.Note: Heat inactivation significantly reduces transformation efficiency due to the presence of PEG in the buffer.4. After the reaction, store the DNA ligation product at 0-4℃, and then carry out transformation experiments; you can also store the DNA ligation product at -20℃.Note: When transforming by chemical method, do not add more than 10% of the volume of the receptor cell for the ligation product.5. Heat shock the ligation product to transform 50 µl of receptor cells or take 1-2 µl of ligation product to electroshock transform 50 µl of receptor cells.Note: 1) When transforming by chemical method, do not add more than 10% of the volume of the receptor cell for the ligation product.(2) If the quick ligation product is used for electrotransformation, it is recommended to use a centrifugal column to purify the ligation product from DNA before electrotransformation because the PEG in the quick ligation reaction system will affect the efficiency of electrotransformation... Read More | This kit is suitable for extracting total RNA from fresh whole blood (blood samples treated with anticoagulants such as citrate, EDTA, or heparin). It can process up to 1.5 ml of whole blood and elute to obtain high-purity RNA with a molecular weight greater than 200 bp. Multiple samples can be This kit is suitable for extracting total RNA from fresh whole blood (blood samples treated with anticoagulants such as citrate, EDTA, or heparin). It can process up to 1.5 ml of whole blood and elute to obtain high-purity RNA with a molecular weight greater than 200 bp. Multiple samples can be completed simultaneously within 1 hour. This product does not require the ultra centrifugation step of CsCl purification and LiCl or ethanol precipitation. It does not contain toxic solvents such as phenol or chloroform. The purified RNA effectively removes enzyme inhibitors and pollutants such as heme and heparin. It can be directly used in various molecular biology routine experiments, such as RT-PCR, Northern Blot, Dot Blot, in vitro translation, and so on.Self prepared reagents: β- Mercaptoethanol, 70% ethanol (prepared with water without RNase), anhydrous ethanol. R666034 Component 50 T Storage R666034A Buffer RBL (10×) 60 mL RT R666034B Buffer RL 35 mL RT R666034C Buffer RW1 40 mL RT R666034D Buffer RW2 (concentrate) 11 mL RT R666034E RNase-Free Water 10 mL RT R666034F Spin Columns FL with Collection Tubes 50 sets RT R666034G Spin Columns RM with Collection Tubes 50 sets RT R666034H RNase-Free Centrifuge Tubes (1.5 mL) 50 EA RT Preparation and important precautions before the experimentTo 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 0.5M 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 sample should avoid repeated freezing and thawing, otherwise it will affect the yield and quality of RNA extraction. The sample can be stored in Buffer RL at -70 ℃ for one month.3. Before use, please check if there is any crystallization or precipitation in the Buffer RL. It can be dissolved again in a 56 ℃ water bath. 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.4. Before the first use, anhydrous ethanol should be added to Buffer RW2 according to the instructions on the reagent bottle label.5. This reagent kit cannot be used for RNA extraction from frozen blood samples with anticoagulants added.6.10 × Buffer RBL needs to be diluted 10 times with water without RNase before use, and then stored at 2-8 ℃ after dilution.7. If downstream experiments are highly sensitive to DNA, it is recommended to treat RNA with DNase I that does not contain RNase.8. All centrifugation steps should be carried out at room temperature unless otherwise specified, and all operation steps should be carried out quickly.Operation steps1. Add 5 times the volume of 1 x Buffer RBL to fresh anticoagulant whole blood samples of 0.5-1.5 ml (please dilute 10 x Buffer RBL with RNase free water before use), gently vortex or invert and mix well. Incubate on ice for 10-15 minutes, mix twice during the incubation process.Attention: During the incubation process, the cloudy suspension will become transparent, indicating that red blood cells have been lysed. If necessary, the incubation time can be extended to 20 minutes. 2. Centrifuge at 4 ℃, 2100 rpm (~400 × g) for 10 minutes, and carefully discard the supernatant.3. Add 2 times the volume of the blood sample to the above precipitate with 1 x Buffer RBL (please dilute 10 x Buffer RBL with RNase free water before use), gently vortex, and resuspend the precipitate thoroughly. 4. Centrifuge at 4 ℃ and 2100 rpm for 10 minutes, carefully and thoroughly remove the supernatant.Note: This step must completely remove the supernatant, otherwise it will affect the lysis and lead to a decrease in RNA production.5. Add Buffer RL to the precipitate (check if it has been added before use β- Mercaptoethanol, 0.5-1.5 ml of blood sample added to 600 µ L Buffer RL, or less than 0.5 ml of blood sample added to 350 µ L Buffer RL, mix well.6. Transfer the obtained liquid to the spin columns FL that have been loaded into the collection tube, centrifuge at 12000 rpm (~13400 × g) for 2 minutes, collect the filtrate, and discard the filter column.7. Add 1 volume (600) to the obtained filtrate µ L or 350 µ l) Mix 70% ethanol (prepared without RNase water) well.Attention: Adding ethanol may cause precipitation and will not affect subsequent experiments.8. Add all the solution obtained in the previous step to the spin columns RM that have been loaded into the collection tube. If the solution cannot be added at once, it can be transferred in multiple batches. 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.9. Add 700 to the adsorption column µ Centrifuge at 12000 rpm for 15 seconds, 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 9 with the following steps.1) Add 350 to the adsorption column µ Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid from the collection tube, and place the adsorption column back into the collection tube.2) Preparation of DNase I mixture: Take 70 µ Reaction Buffer and 10 µ L DNase I storage solution, gently mix 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 (D665537) reaction system. Please refer to the corresponding manual for other company products.1) Add 350 to the adsorption column µ Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid from the collection tube, and place the adsorption column back into the collection tube.2) Preparation of DNase I mixture: Take 70 µ Reaction Buffer and 10 µ L DNase I storage solution, gently mix 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 (D665537) reaction system. Please refer to the corresponding manual for other company products.3) Add 80 µ l of the prepared 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 Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid from the collection tube, and place the adsorption column back into the collection tube.10. Add 500 to the adsorption column µ Buffer RW2 (check if anhydrous ethanol has been 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.11. Repeat step 10. 12. Centrifuge at 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 air dry.Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).13. Place the adsorption column in a new RNase free centrifuge tube and add 30-50 to the middle of the adsorption column 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 13 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 13... Read More | Product contentS666146Component50 T200 TStorageS666146ABuffer GR25 mL120 mLRTS666146BBuffer GL25 mL120 mLRTS666146CBuffer GW1 (concentrate)13 mL52 mLRTS666146DBuffer GW2 (concentrate)15 mL75 mLRTS666146EBuffer GE15 mL60 mLRTS666146FProteinase K1.25 mL4×1.25 mLRTS666146GSpin Columns DS with Product contentS666146Component50 T200 TStorageS666146ABuffer GR25 mL120 mLRTS666146BBuffer GL25 mL120 mLRTS666146CBuffer GW1 (concentrate)13 mL52 mLRTS666146DBuffer GW2 (concentrate)15 mL75 mLRTS666146EBuffer GE15 mL60 mLRTS666146FProteinase K1.25 mL4×1.25 mLRTS666146GSpin Columns DS with Collection Tubes50 sets 200 setsRTS666146HCentrifuge Tubes (1.5 mL)50 EA200 EARTProductsThis kit provides a simple and rapid method for the isolation and purification of total DNA from buccal swab samples. The kit adopts a silica matrix membrane that can specifically bind DNA and a unique buffer system to adsorb DNA efficiently and specifically, and 0.5-3.5 µg of genomic DNA can be obtained from each swab, and the extracted DNA fragments are large, pure and of stable and reliable quality. It is suitable for enzyme digestion, PCR, library construction, Southern hybridization and other experiments.Self-contained reagent: anhydrous ethanol.Pre-experiment Preparation and Important Notes1. Anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the instructions on the label of the reagent bottle before first use.2. If precipitation is found in Buffer GL before use, dissolve Buffer GL in a 56°C water bath.3. All centrifugation steps can be performed at room temperature.4. Sampling: Use a buccal swab to wipe the inside of the mouth 6 times, dry for 2 hours and store. To ensure that the sample is not contaminated by food or drink, do not eat or drink for 30 minutes before sampling.Procedure1. The swab of the buccal swab was cut from the rod with scissors and placed in a 2mL centrifuge tube (supplied) and 400µL Buffer GR was added.Note: For genomic DNA without RNA contamination, add 4 µL of RNase A solution at a concentration of 100 mg/ml and shake to mix.2. Add 20 µL of Proteinase K and 400 µL of Buffer GL, immediately vortex and shake for 15 seconds and mix thoroughly.Note: Mix well immediately after adding Buffer GL; do not add Proteinase K directly to Buffer GL for use.3.56°C for 10 minutes and centrifuge briefly so that the solution on the walls of the tube collects at the bottom.4. Add 400 µL of anhydrous ethanol, vortex and shake to mix thoroughly, and centrifuge briefly so that the solution on the wall of the tube collects at the bottom of the tube.Note: The addition of anhydrous ethanol may produce a white precipitate that will not affect subsequent experiments.5. Add the solution and precipitate obtained in the previous step to the Spin Columns DS in two batches of up to 700 µL at a time into the collection tube. centrifuge the column at 12,000 rpm (∼13,400 × g) for 1 minute, pour off the waste liquid from the collection tube, and return the column to the collection tube.6. Add 500 µL of Buffer GW1 to the adsorbent column (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the adsorbent column back into the collection tube.7. Add 500 µL of Buffer GW2 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge the column at 12,000 rpm for 3 minutes, pour off the waste liquid in the collection tube, and put the column back into the collection tube.Note: Step 7 can be repeated if further DNA purity is required.8. Centrifuge at 12,000 rpm for 1 minute and pour off the waste liquid in the collection tube. Leave the adsorption column at room temperature for several minutes to dry thoroughly.Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can interfere with subsequent enzymatic reactions (digestion, PCR, etc.).9. Place the adsorption column in a new 1.5 mL centrifuge tube, add 50 µL of Buffer GE or sterilized water to the middle of the adsorption column overhanging the column, let stand at room temperature for 2-5 minutes, centrifuge at 12,000 rpm for 1 minute, collect the DNA solution, and store at -20℃.Attention:(1) If the downstream experiment is sensitive to pH or EDTA, it can be eluted with sterilized water. The pH value of the eluent has a great influence on the elution efficiency. If the eluent is made of water, the pH value should be 7.0-8.5 (the pH value of water can be adjusted to this range by using NaOH), and the elution efficiency is not high when the pH value is lower than 7.0.2) For long-term storage, it is recommended to elute with Buffer GE and store at -20°C... Read More |