| Description | Inquire | B669951 Component 50T Storage B669951A Buffer ATL 15 mL RT B669951B Buffer AL 15 mL RT B669951C Buffer AW1 (concentrate) 13 mL RT B669951D Buffer AW2 (concentrate) 15 mL RT B669951E Buffer EB 15 mL RT B669951F Proteinase K 1.25 mL RT B669951G Spin Columns DM with Collection Tubes 50 sets B669951 Component 50T Storage B669951A Buffer ATL 15 mL RT B669951B Buffer AL 15 mL RT B669951C Buffer AW1 (concentrate) 13 mL RT B669951D Buffer AW2 (concentrate) 15 mL RT B669951E Buffer EB 15 mL RT B669951F Proteinase K 1.25 mL RT B669951G Spin Columns DM with Collection Tubes 50 sets RTProductsThis kit is suitable for extracting high purity total DNA from Gram-negative and Gram-positive bacteria. 106-108 cells can be processed at a time, and up to 20 µg of total DNA can be obtained within one hour without the need for toxic solvents such as phenol or chloroform, and without the need for ethanol precipitation. The optimized buffer system enables the DNA in the lysate to be efficiently and specifically bound to the silica matrix centrifugal adsorption column, while other contaminants can flow through the membrane, and the inhibitors of PCR and other enzymatic reactions can be effectively removed through a two-step washing step, and finally washed off with low-salt buffer or water, so that high-purity DNA can be obtained.The purified DNA can be used for downstream experiments such as digestion, PCR, Real-Time PCR, library construction, Southern Blot and molecular labeling, molecular labeling and other downstream experiments. Self-contained reagents: anhydrous ethanol; Enzymatic Lysis Buffer is required for extraction of Gram-positive bacteria.Enzymatic Lysis Buffer was prepared by 20 mM Tris, pH 8.0; 2 mM Na2-EDTA, pH 8.0; and 1.2% Triton X-100. 121°C sterilization for 20 minutes, and the appropriate amount of Lysozyme was added at a final concentration of 20 mg/ml. Pre-experiment Preparation and Important Notes1. Add 1.25ml Proteinase K Storage Buffer to Proteinase K to dissolve it and store it at -20℃. Do not leave the prepared Proteinase K at room temperature for a long time, and avoid repeated freezing and thawing to avoid affecting its activity.2. Repeated freezing and thawing of the sample should be avoided, as this may result in smaller DNA fragments and a decrease in the amount of extracted DNA.3. If extracting genomes from bacterial cultures with high accumulation of secondary metabolites or thick cell walls, it is recommended that samples be collected early in the logarithmic phase.4. 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.5. Before use, please check Buffer GTL and Buffer GL for crystallization or precipitation. If crystallization or precipitation occurs, please re-dissolve Buffer GL and Buffer GTL in a 56℃ water bath.6. If the downstream experiments are sensitive to RNA contamination, 4µl of DNase-Free RNase A (100mg/ml) can be added before adding Buffer GL. RNase A is not provided in this kit.If the extracted samples are Gram-positive bacteria, customers need to prepare their own Enzymatic Lysis Buffer to treat the bacteria, which requires the use of Lysozyme (lysozyme) at a concentration of 20 mg/ml, which is not provided in this kit.Procedurei Extraction of genomic DNA from Gram-negative bacteria1. Take 1-5 ml of bacterial culture (106-108 cells, maximum 2×109 cells) and put it into a centrifuge tube (provided), centrifuge it at 12,000 rpm (~13,400×g) for 1 minute, and aspirate the supernatant as much as possible.2. Add 180 µl Buffer GTL to the precipitate and shake to resuspend the bacteria.3. Add 20 µl of Proteinase K, vortex and mix well, incubate at 56°C until the solution becomes clear, and invert or shake the centrifuge tube at intervals during the incubation to disperse the sample.Note: If RNA removal is required, add 4 µl of RNase A solution at a concentration of 100 mg/ml after the above steps are completed, shake to mix, and leave for 5-10 minutes at room temperature.4. Add 200µl Buffer GL and mix well with vortexing and shaking. Add 200µl of anhydrous ethanol and mix well with vortexing and shaking.Centrifuge briefly so that the solution on the walls of the tube collects at the bottom.Note: 1) If multiple samples are manipulated together, Buffer GL and anhydrous ethanol can be mixed in equal proportions and then added together, shaking to mix.2) The addition of Buffer GL and anhydrous ethanol may produce a white precipitate that will not affect subsequent experiments.5. Add all of the solution obtained in step 4 (including the precipitate formed) to the Spin Columns DM in the collection tube, or if the solution cannot be added all at once, transfer it several times. centrifuge at 12,000 rpm for 1 minute, discard the waste solution, and return the column to the collection tube.6. Add 500 µl of Buffer GW1 to the adsorption 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 return the adsorption column to the collection tube.7. Add 500 µl of Buffer GW2 to the adsorption 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 adsorption 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 2 minutes and pour off the waste liquid in the collection tube. Leave the adsorbent column at room temperature for several minutes to dry thoroughly. Note: The purpose of this step is to remove residual ethanol from the adsorbent column; ethanol residue can interfere with subsequent enzymatic reactions (digestion, PCR, etc.).9. Place the adsorption column in a new centrifuge tube, add 50-200 µl Buffer GE to the middle part of the adsorption column overhanging the center of the adsorption column, leave it at room temperature for 2-5 minutes, centrifuge it at 12,000 rpm for 1 minute, collect the DNA solution, and store the DNA at -20 ℃. note: 1) If the downstream experiments are sensitive to the pH or EDTA, the elution can be done with sterilized water. The pH of the elution solution has a great influence on the elution efficiency. If water is used as the elution solution it should be ensured that its pH is 7.0-8.5 (the pH of water can be adjusted to this range with NaOH), and the elution efficiency is not high when the pH is lower than 7.0.2) Incubation at room temperature for 5 minutes prior to centrifugation increases yield.3) Re-elution with an additional 50-200 µl Buffer GE or sterilized water can increase the yield.4) If the final concentration of DNA is to be increased, the DNA eluate obtained in step 9 can be re-spiked onto the adsorbent membrane and step 9 repeated; if the elution volume is less than 200 µl, the final concentration of DNA can be increased, but the total yield may be reduced. If the amount of DNA is less than 1 µg, elution with 50 µl Buffer GE or sterilized water is recommended.(5) DNA stored in water will be affected by acidic hydrolysis. For long-term storage, it is recommended to elute with Buffer GE and store at -20℃.i. Extraction of genomic DNA from Gram-positive bacteria1. Take 1-5 ml of bacterial culture (106-108 cells, maximum 2×109 cells) and put it into a centrifuge tube (provided), centrifuge it at 12,000 rpm (~13,400×g) for 1 minute, and aspirate the supernatant as much as possible.2. Add 180µl Enzymatic Lysis Buffer (self-provided) to resuspend the bacteria.Enzymatic Lysis Buffer is prepared as described in the Self-Prepared Reagents section in the front of the manual.3. Incubate at 37°C for 30 minutes.4. Add 20µl Proteinase K and mix well. Add 200µl of Buffer GL and mix well with vortexing and shaking.Note: Do not add Proteinase K directly to Buffer GL.Incubate at 5.56°C for 30 minutes.Note: 1) If desired, incubation at 95°C for 15 minutes will inactivate the pathogen, but 95°C incubation will cause some DNA degradation.(2) If RNA removal is required, add 4µl of RNase A solution at a concentration of 100mg/ml after the above steps are completed, shake and mix well, and leave for 5-10 minutes at room temperature.6. Add 200µl of anhydrous ethanol and mix well with vortex shaking.Note: The addition of anhydrous ethanol may produce a white precipitate that will not affect subsequent experiments.7. Add all of the solution obtained in step 6 (including the precipitate formed) to the Spin Columns DM that have been loaded into the collection tube, and if the solution cannot be added all at once, it can be transferred in several times. centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid from the collection tube, and put the column back into the collection tube.8. Add 500 µl of Buffer GW1 to the adsorption 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 adsorption column back into the collection tube.9. Add 500 µl Buffer GW2 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge the column at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the column back into the collection tube.Note: Step 9 can be repeated if further DNA purity is required.10. Centrifuge at 12,000 rpm for 2 minutes 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; ethanol residue can interfere with subsequent enzymatic reactions (digestion, PCR, etc.).11. Place the adsorption column in a new centrifuge tube (self-provided), add 50-200 µl of Buffer GE to the center of the adsorption column overhanging the center of the adsorption column, let it stand at room temperature for 2-5 minutes, centrifuge at 12,000 rpm for 1 minute, collect the DNA solution, and store the DNA at -20℃.Note: 1) If the downstream experiment is sensitive to pH or EDTA, you can use sterilized water for elution. The pH of the eluent has a great influence on the elution efficiency, if water is used as the eluent should ensure that its pH is 7.0-8.5 (you can use NaOH to adjust the pH of the water to this range), and the elution efficiency is not high when the pH is lower than 7.0.2) Incubation at room temperature for 5 minutes prior to centrifugation increases yield.3) Re-elution with an additional 50-200 µl Buffer GE or sterilized water can increase the yield.4) If the final concentration of DNA is to be increased, the DNA eluate obtained in step 11 can be re-spiked onto the adsorbent membrane and step 11 repeated; if the elution volume is less than 200 µl, the final concentration of DNA can be increased, but the total yield may be reduced. If the amount of DNA is less than 1 µg, elution with 50 µl Buffer GE or sterilized water is recommended.(5) DNA stored in water will be affected by acidic hydrolysis. For long-term storage, it is recommended to elute with Buffer GE and store at -20℃... Read More | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | Hydrogen peroxide, a reactive oxygen species produced through the metabolism of molecular oxygen, serves as both an intracellular signaling messenger and a source of oxidative stress. Hydrogen peroxide is generated in cells via multiple mechanisms such as the NOX-mediated ROS production by Hydrogen peroxide, a reactive oxygen species produced through the metabolism of molecular oxygen, serves as both an intracellular signaling messenger and a source of oxidative stress. Hydrogen peroxide is generated in cells via multiple mechanisms such as the NOX-mediated ROS production by neutrophils and macrophages (respiratory burst) or by the dismutase of superoxide anions produced as a result of electron leak during mitochondrial respiration. Abnormal hydrogen peroxide production contributes to oxidative cell damage and the progression of diseases such as asthma, atherosclerosis, osteoporosis, and neurodegeneration.Intracellular hydrogen peroxide assay kit has been used to measure intracellular hydrogen peroxide levels... Read More | Product contentP666142Component200 TStorageP666142ABuffer P160 mLRTP666142BBuffer P260 mLRTP666142CBuffer N380 mLRTP666142DBuffer PB35 mLRTP666142EBuffer PW (concentrate)25 mLRTP666142FBuffer EB30 mLRTP666142GRNase A (10 mg/mL)600 µLRTP666142HSpin Columns DM with Collection Tubes200 EART Product contentP666142Component200 TStorageP666142ABuffer P160 mLRTP666142BBuffer P260 mLRTP666142CBuffer N380 mLRTP666142DBuffer PB35 mLRTP666142EBuffer PW (concentrate)25 mLRTP666142FBuffer EB30 mLRTP666142GRNase A (10 mg/mL)600 µLRTP666142HSpin Columns DM with Collection Tubes200 EART Product IntroductionThis kit is suitable for extracting 1-5 ml of bacterial solution. Based on the lysis of cells by alkaline lysis method, it adopts a unique silica matrix membrane adsorption technology and reagent formulation, and efficiently and exclusively binds plasmid DNA in solution by centrifugal adsorption columns in a high-salt state, and each adsorption column can adsorb a maximum of 30 µg of plasmid DNA, and removes proteins, genomes, RNAs, and other impurities to the greatest extent possible. The plasmid DNA obtained can be directly used for cell transfection, PCR, digestion, sequencing, ligation and other biological experiments.Self-contained reagent: anhydrous ethanol.Pre-experiment Preparation and Important Notes1. All components can be stably stored in dry, room temperature (15-30℃) environment for 1 year, the adsorption column can be stored at 2-8℃ for a longer period of time, and Buffer P1 with RNase A can be stably stored at 2-8℃ for 6 months.2. Before the first use, add all the RNase A solution into Buffer P1, mix well, and store it at 2-8°C. Before use, leave it at room temperature for a period of time, and then use it after recovering to room temperature.3. Anhydrous ethanol should be added to Buffer PW according to the instructions on the label of the reagent bottle before first use.4. If precipitation is found in Buffer P2, Buffer N3, or Buffer PB before use, the clarification can be restored by water bath at 37℃ for a few minutes (please do not shake Buffer P2 violently).5. Be careful not to touch Buffer P2, Buffer N3 and Buffer PB directly, and tighten the lid immediately after use.6. The amount and purity of extracted plasmid is related to the concentration of bacterial culture, strain type, plasmid size, plasmid copy number and other factors.Procedure1. Take 1-5 ml of the overnight culture and add it to a centrifuge tube (self-prepared), centrifuge for 30 seconds at 13,000 rpm (~16,200×g) to collect the bacterial precipitate, and discard the supernatant as much as possible.2. Add 250 µl of Buffer P1 to the centrifuge tube with the bacterial precipitate (please check that RNase A has been added first), mix well using a pipette or vortex shaker, and suspend the bacterial precipitate.Note: If the bacterial mass is not thoroughly mixed, it will affect the lysis effect, resulting in low extraction and purity.3. Add 250µl of Buffer P2 to the centrifuge tube and mix gently up and down 4-6 times, mixing well to lyse the organisms, at which point the solution should become clear and viscous.Note: Mix gently, do not shake vigorously to avoid interrupting the genomic DNA and causing the extracted plasmid to be mixed with genomic DNA fragments. This step should take no more than 5 minutes to avoid damage to the plasmid.4. Add 350 µl of Buffer N3 to the centrifuge tube and immediately mix gently up and down for 8-10 times, mixing well so that a white flocculent precipitate should appear. centrifuge at 13,000 rpm for 5 minutes.Note: Buffer N3 should be mixed immediately after addition to avoid localized precipitation.5. Transfer the supernatant obtained in step 4 to the Spin Columns DM that have been loaded into the collection tube, centrifuge at 13,000 rpm for 30 seconds, pour off the waste liquid from the collection tube, and place the column back into the collection tube.6. Add 150 µl Buffer PB to the adsorption column and centrifuge at 13,000 rpm for 30 seconds.7. Add 400 µl Buffer PW to the adsorption column (please check that anhydrous ethanol has been added first), centrifuge at 13,000 rpm for 1 minute, and pour off the waste liquid in the collection tube.8. Place the adsorbent column in a new centrifuge tube (supplied), add 50-100 µl Buffer EB to the middle of the adsorbent membrane, leave it at room temperature for 2 minutes, centrifuge at 13,000 rpm for 1 minute, and collect the plasmid solution into the centrifuge tube. -The plasmid solution was collected into the centrifuge tube.Note: 1) To increase the recovery efficiency of the plasmid, the resulting solution can be reintroduced into the adsorbent column, left at room temperature for 2 minutes, centrifuged at 13,000 rpm for 1 minute, and the plasmid solution collected into a centrifuge tube.2) For low plasmid copy number or >10 kb, Buffer EB is preheated at 65-70°C in a water bath to increase extraction efficiency... Read More |