| Description | Inquire | Annexin V ( annexin-V ) is a Ca2 + dependent phospholipid binding protein with a molecular weight of 35-36 KD, which can selectively bind to phosphatidylserine ( PS ). Phosphatidylserine ( PS ) is mainly distributed in the inner side of the cell membrane, that is, the side adjacent to the cytoplasm.Annexin V ( annexin-V ) is a Ca2 + dependent phospholipid binding protein with a molecular weight of 35-36 KD, which can selectively bind to phosphatidylserine ( PS ). Phosphatidylserine ( PS ) is mainly distributed in the inner side of the cell membrane, that is, the side adjacent to the cytoplasm. In the early stage of apoptosis, different types of cells will turn phosphatidylserine out to the cell surface and expose to the extracellular environment. At this time, using Annexin V labeled with fluorescent protein PE, that is, Annexin V-PE, combined with phosphatidylserine ( PS ), the eversion of phosphatidylserine, an important feature of apoptosis, can be directly detected by flow cytometry. Normal cells will not be stained by Annexin V-PE, apoptotic or necrotic cells will be stained by Annexin V-PE. Annexin V-PE can be used in combination with partially non-permeable nuclear dye ( 7-AAD / PI ) to distinguish cells at different stages of apoptosis. RedNucleus II provided in this kit is a far-red dye that belongs to an anthraquinone compound and cannot penetrate the intact cell membrane of living cells and early apoptotic cells. It is non-permeable, but can quickly stain the nucleus / dsDNA in dead and permeable cells. RedNucleus II is an ideal substitute for propidium iodide ( PI ) and 7-AAD.Combined with Annexin V-PE, it has better spectral characteristics without compensation regulation : it is not excited by ultraviolet light and does not overlap with PE / PE homologues, so it can be combined with FITC, PE and purple fluorescent dyes for multicolor analysis. When combined with Annexin V-PE, RedNucleus II was excluded from living cells and early apoptotic cells, while late apoptotic cells and dead cells were double-positive for Annexin V-PE and RedNucleus II. Annexin V-PE / RedNucleus II apoptosis detection kit can be detected by flow cytometry or other fluorescence detection equipment. Components: Components A598354(10T) A598354(50T) A598354(100T) A. 1×Annexin V Combining buffer solution 10 mL 50 mL 50 mL×2 B. Annexin V-PE 50 µL 250 µL 500 µL C. RedNucleus II 100 µL 500 µL 1 mLProduct parameters：Annexin v-pe:ex/em=488/578 nmrednucleus ii:ex/em=635/695 NMUsage method:1. Experimental design: Blank tube: Negative control group cells, without Annexin V-PE/RedNucleus II. Used to regulate voltage.Single staining tube: Positive control group cells were treated with Annexin V-PE alone/RedNucleus II alone. Used for adjusting compensation.Detection tube: Add Annexin V-PE/RedNucleus II to the processed cells. After adjusting the voltage compensation using blank tubes and single dye tubes, obtain the required flow data.2. Collect cells(1) For suspended cells:a. After inducing cell apoptosis, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, collect the cells, gently resuspend the cells in PBS, and count them.Note: PBS resuspension cannot be omitted. The process of PBS resuspension also serves to wash cells, ensuring the subsequent binding of Annexin V-PE.b. Take 5 × 104-1 × 105 resuspended cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add 100 µ L of 1 × Annexin V binding buffer to gently resuspend the cells. c. Add 5 µ L Annexin V-PE and mix gently.d. Add 5 µ L of RedNucleus II staining solution and mix gently.e. Incubate at room temperature (20-25 º C) in the dark for 15 minutes. Aluminum foil can be used to avoid light. During the incubation process, cells can be resuspended 2-3 times to improve staining efficiency.(2) For adherent cells:a. Suck out the cell culture medium into a suitable centrifuge tube, wash the adherent cells with PBS once, and add an appropriate amount of trypsin cell digestion solution (without EDTA) to digest the cells. Incubate at room temperature until gently blowing can remove the trypsin cell digestion solution when the adherent cells are blown down. Overdigestion of pancreatic enzymes should be avoided.Note: For adherent cells, the trypsin digestion step is crucial. If the trypsin digestion time is too short, cells need to be blown hard to detach, which can easily cause damage to the cell membrane and lead to false positives of cell necrosis; If the digestion time is too long, it can also cause cell membrane damage and false positives of cell necrosis, and even affect the binding of phosphatidylserine and Annexin V-PE on the cell membrane, thereby interfering with the detection of cell apoptosis.b. Add the cell culture medium collected in the previous step, gently blow down the cells, transfer them to a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, collect the cells, gently resuspend the cells in PBS and count them.Note: Adding the cell culture medium from the previous step is very important. On the one hand, it can collect cells that have already been suspended and undergone apoptosis or necrosis. On the other hand, the serum in the cell culture medium can effectively inhibit or neutralize residual trypsin. The residual trypsin will digest and degrade the subsequently added Annexin V-PE, leading to staining failure.c. Take 5 × 104-1 × 105 resuspended cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add 100 µ L of 1 × Annexin V binding buffer to gently resuspend the cells. d. Add 5 µ L Annexin V-PE and mix gently.e. Add 5 µ L of RedNucleus II staining solution and mix gently.f. Incubate at room temperature (20-25 º C) in the dark for 15 minutes. Aluminum foil can be used to avoid light. During the incubation process, cells can be resuspended 2-3 times to improve staining efficiency.3. Result analysis:(1) Flow cytometry detection:a. After incubation, 400 µ L of 1 × Annexin V binding buffer can be directly added to resuspend the cells, and immediately detected on the machine. Annexin V-PE is excited by 488 nm/566 nm laser, and the fluorescence emission spectrum is detected at 578 nm (BL2 (FL2)/YL1 channel), while the RedNucleus II channel emission spectrum is approximately at 695 nm (RL1 (FL4) channel).b. On the scatter plot of the bivariate flow cytometer, live cells are shown in the lower left quadrant, which is (Annexin V-PE -/RedNucleus II -); The lower right quadrant represents early apoptotic cells, which are (Annexin V-PE+/RedNucleus II -); The upper right quadrant represents necrotic and late stage apoptotic cells, which are (Annexin V-PE+/RedNucleus II+); The upper left quadrant displays naked nuclear cells, which are (Annexin V-PE -/RedNucleus II+).(2) Fluorescence microscopy detection:a. Centrifuge at 1000 rpm for 5 minutes, collect cells, and gently resuspend them in 400 µ L of 1 × Annexin V binding buffer. Transfer the cells to a 96 well plate and settle for a moment or perform cell smear, then observe under a fluorescence microscope.b. Annexin V-PE is compatible with PE filters. RedNucleus II can use a far red long pass filter.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. to reduce the process of apoptosis, the incubation process can be operated on ice, but the incubation time should be extended to at least 30 min. 3. as apoptosis is a rapid process, it is recommended that samples be analyzed within 1 h after staining. 4. for adherent cells, digestion is a key step. If there are floating cells when adherent cells induce apoptosis, the floating cells and adherent cells should be collected and stained. Handle adherent cells with care to avoid artificial damage to cells. The trypsin digestion time is too short, and the cells need to be blown hard to fall off, which is easy to cause damage to the cell membrane and excessive intake of rednucleus II; If the digestion time is too long, the cell membrane is also prone to damage, and even affect the binding of phosphatidylserine and annexin v-pe on the cell membrane. When digesting, spread pancreatin on the bottom of the well plate, fully contact the pancreatin with the cells when shaking gently, then pour out most of the pancreatin, use the remaining small amount of pancreatin to digest for a period of time, and terminate when the gap between cells increases and the bottom of the bottle is spotted. Try not to use EDTA in the digestive juice, which will affect the binding of annexin V to PS. 5. after the adherent cells are digested with trypsin, it is recommended to stain after recovering in the optimal culture conditions and medium for about 30 min to avoid false positives. 6. in order to avoid losing cells when washing cells, you can use a large tip over a small tip to aspirate. 7. the optimal concentration of dye is determined by the specific experimental requirements. 8. fluorescent dyes have quenching problems. Please try to avoid light during storage and use to slow down fluorescence quenching. 9. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Early apoptosis detection, annexin V Kit... Read More | Products content Products IntroductionThis kit is a dedicated sample preparation solution for microbiome analysis and is suitable for the purification and enrichment of genomic DNA of pathogenic microorganisms such as bacteria and fungi from mixed samples such as swabs, blood, sputum, alveolar Products content Products IntroductionThis kit is a dedicated sample preparation solution for microbiome analysis and is suitable for the purification and enrichment of genomic DNA of pathogenic microorganisms such as bacteria and fungi from mixed samples such as swabs, blood, sputum, alveolar lavage, etc. During the purification process, differential lysis of the host cells and subsequent enzymatic digestion can effectively remove most of the host DNA while providing a comprehensive coverage of the bacterial and fungal DNA loci to a higher level. By differential lysis of host cells and subsequent enzymatic digestion, this kit can effectively remove most of the host DNA while maximizing the full coverage of bacterial, fungal and other pathogenic microbial DNA sites, thus obtaining microbiome DNA enrichment products with a higher coverage. Microbial DNA purified with this kit is suitable for a variety of downstream applications, including whole genome sequencing analysis, 16S rDNA-based high sensitivity microbiome analysis, and macrogenomic birdshot sequencing analysis. Self-contained reagents and consumablesSterile pipette tips with aerosol barrier to prevent cross-contamination anhydrous ethanol Microcentrifuge tubes (2 ml/1.5 ml) PBS buffer (required for some samples only)Pre-experiment Preparation and Important Notes1. Add 1.25 ml Proteinase K Storage Buffer to Proteinase K and store at -20℃. Do not leave the prepared Proteinase K (20 mg/ml) at room temperature for a long time, and avoid repeated freezing and thawing to avoid affecting its activity.2. Dissolve Lysozyme (100 mg) in 10 ml Enzymatic Lysis Buffer to a final concentration of 10 mg/ml, dispense into sterile tubes and store at -20℃. Do not leave the prepared Lysozyme (10 mg/ml) at room temperature for a long time and avoid repeated freezing and thawing to avoid affecting its activity.3. Thaw Buffer GB1 and Buffer GB2 at room temperature or 2-8°C before use and mix thoroughly. Thawed Buffer GB1 and Buffer GB2 can be left at 2-8°C for 1-2 weeks without affecting their activity, and should be stored at -20°C for long term storage. To ensure optimal performance, do not freeze or thaw more than three times. If less than one bottle of Buffer GB1 and Buffer GB2 is required for a single extraction, ensure that it is used under sterile conditions such as an ultra-clean bench and avoid microbial contamination and growth in the remaining buffer.4. Before first use, anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the instructions on the vial label and labeled.5. Check Buffer GL for crystallization or precipitation before use, and if crystallization or precipitation occurs, redissolve Buffer GL in a 56°C water bath.6. If the downstream experiments are sensitive to RNA contamination, 4 µl of DNase-Free RNase A (100 mg/ml) can be added before adding Buffer GL. RNase A is not provided in the kit, but can be ordered separately from CW0601S.7. This kit is designed for the isolation of DNA from intact microbial cells. To ensure optimal recovery of microbial DNA, samples should be fresh. If storage or transportation is required, this should preferably be done at 2-8°C and not frozen or thawed, as freezing and thawing can damage the integrity of the microbial cells and therefore result in the loss of exposed microbial DNA during host DNA removal.8. To avoid false results due to contamination, keep the work area clean, wear protective clothing, and set up controls for quality control. Use appropriate measures to handle sample materials to minimize the risk of cross-contamination. During the extraction process, use DNA-free pipette tips and consumables, and cap reagents immediately after use to prevent contamination. procedure1. Sample pre-treatment: 1a: For swab samples, swirl the swab portion of the swab in 0.5 ml PBS for at least 20 s. Squeeze the swab several times against the wall of the tube before removing it so that as much of the bacterial fluid as possible can be squeezed out of the swab to minimize sample loss. 1b: For viscous samples, e.g. sputum, take ~500 µl of sample, add 1.5 times the volume (~750 µl) of Buffer GB1 and incubate at 37°C, 600 rpm for 15-30 min until the sample is completely liquefied.Note: The sample volume can be increased or decreased appropriately and the amount of Buffer GB1 added adjusted accordingly.1c: For alveolar lavage fluid containing a small amount of viscous sputum, centrifuge as much of the alveolar lavage fluid as possible, carefully remove the supernatant, and retain the lower viscous fraction (containing sputum, cells, and organisms), add 1.5 times the volume of Buffer GB1, and incubate for 15-30 min at 37°C, 600 rpm until the sample is completely liquefied.1d: For non-viscous body fluid samples such as blood and cerebrospinal fluid, liquefaction treatment is not required, and an appropriate amount of sample is taken directly, the operation of step 2 is carried out, and the cell precipitate is collected by centrifugation.2. Centrifuge at 10000 rpm for 5-10 min at room temperature and carefully discard the supernatant.Note: Do not disturb the lower cell sediment to avoid sample loss.3. Add 500 µl Buffer GB2, vortex to mix, and incubate at room temperature, 600 rpm for 10 min. 4. Centrifuge at 12000 rpm for 2 min and carefully remove the supernatant.Note: Do not disturb the bacterial precipitate when removing the supernatant to avoid sample loss.5. Add 200 µl of Buffer GB2 to the precipitate, add 2 µl of Benzonase and incubate for 30 min at 37°C, 600 rpm. 6. Centrifuge at 12000 rpm for 2 min, discard the supernatant, add 500 µl of Buffer GB2, vortex and wash the precipitate. Repeat the procedure once.7. Centrifuge at 12000 rpm for 2 min, discard the supernatant, and finally aspirate the residual Buffer GB2 with a small-volume tip. 8. Add 180 µl Lysozyme (10 mg/ml), resuspend the bacterial precipitate and transfer the bacterial resuspension to a Lysis Tube.9. The Lysis Tube is incubated at 37°C, 600 rpm for 20-30 min, then vortexed for 10 min or processed on a thermostatic homogenizer for 10 min at maximum vibration speed (2500-2900 rpm).10. Centrifuge briefly, add 20 µl proteinase K, vortex to mix, add 200 µl buffer GL, vortex to mix, and incubate for 30 min at 56°C, 600 rpm. Note: 1) Do not add Proteinase K directly to Buffer GL.2）For RNA removal, add 4 µl DNase-Free RNase A (100 mg/ml) before adding Buffer GL, shake to mix, and let stand at room temperature for 5-10 minutes.11. Centrifuge at 12000 rpm for 1 min and carefully aspirate the supernatant into a new centrifuge tube. Note: Do not aspirate the glass beads.12. Add 200 µl of anhydrous ethanol, vortex to mix, and centrifuge momentarily to collect the solution to the bottom of the tube. Note: The addition of anhydrous ethanol may produce a white precipitate that will not affect subsequent experiments.13. Add all of the solution from step 12, including the precipitate, to the Spin Columns DM in the collection tube, or transfer the solution several times if it cannot be added all at once. centrifuge at 12,000 rpm for 1 minute, pour off the waste from the collection tube, and return the column to the collection tube.14. Add 500 µl Buffer GW1 to the adsorbent column (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 min, pour off the waste liquid from the collection tube, and put the adsorbent column back into the collection tube.15. Add 500 µl Buffer GW2 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. Note: Step 15 can be repeated once if further improvement of DNA purity is required.16. Centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in the collection tube. Leave the column at room temperature for a few minutes and 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.).17. Place the adsorbent column in a new centrifuge tube (supplied), add 50 µl of Buffer GE to the center of the adsorbent column overhang, let stand at room temperature for 5 minutes, centrifuge at 12,000 rpm for 1 minute, collect the DNA solution, and store the DNA at -20 °C. Attention:1）If the downstream experiments are sensitive to pH or EDTA, sterilized water can be used for elution. 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 with NaOH), and the elution efficiency is not high when the pH value is lower than 7.0.2）Incubation at room temperature for 5 minutes prior to centrifugation increases yield.3）If the final concentration of DNA is to be increased, the DNA eluate obtained in step 17 can be re-spiked onto the adsorbent membrane and step 17 repeated. 4）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 | Product Content R669990Component50 TStorageR669990ADNase I1000 U-20℃. Avoid freeze/thaw cycle.R669990B10×Reaction Buffer1 mL-20℃. Avoid freeze/thaw cycle.R669990CBuffer RL35 mLRTR669990DBuffer RW135 mLRTR669990EBuffer RW2 (concentrate)11 mLRTR669990FRNase-Free Water10 Product Content R669990Component50 TStorageR669990ADNase I1000 U-20℃. Avoid freeze/thaw cycle.R669990B10×Reaction Buffer1 mL-20℃. Avoid freeze/thaw cycle.R669990CBuffer RL35 mLRTR669990DBuffer RW135 mLRTR669990EBuffer RW2 (concentrate)11 mLRTR669990FRNase-Free Water10 mLRTR669990GSpin Columns RM with Collection Tubes50 setsRTR669990HRNase-Free Centrifuge Tubes (1.5 mL)50 EART ProductsThis kit combines highly efficient guanidine isothiocyanate cleavage technology with silica matrix membrane purification for the efficient extraction of total RNA from animal cells and tissues, typically up to 30 mg of tissue or 1x107 cells as a starting sample. The kit also allows recovery of incompletely purified RNA, in vitro transcription and RNA from enzymatic reactions. high quality RNA with molecular weights greater than 200 bases can be extracted and purified using the kit with virtually no DNA residue. If RNA experiments that are very sensitive to trace DNA are to be performed, residual DNA can be removed by on-column digestion using RNase-free DNase. The extracted RNA can be used in downstream experiments such as RT-PCR, Nothern Blot and Dot Blot. Self-contained reagents: β-mercaptoethanol, anhydrous ethanol (freshly opened or for RNA extraction).Pre-experiment Preparation and Important Notes1. To prevent RNase contamination, attention should be paid to the following aspects:1) Use RNase-free plastics and tips to avoid cross-contamination.2) RNase-free water should be used to prepare the solution.(3) Operators wear disposable masks and gloves, and change gloves diligently during the experiment.2. Avoid repeated freezing and thawing of the extracted samples, otherwise it will affect the amount and quality of RNA extraction.3. Please add β-mercaptoethanol to Buffer RL before use, add 10µl of β-mercaptoethanol to 1ml of Buffer RL. Buffer RL with β-mercaptoethanol can be stored for 1 month at room temperature.4. Anhydrous ethanol should be added to Buffer RW2 before first use according to the instructions on the reagent bottle label.5. Buffer RL may be heated at 56°C to dissolve if precipitation occurs and then left at room temperature.All centrifugation steps are performed at room temperature and all maneuvers are performed quickly.Procedure1. Sample handling1a Tissue: Grind tissue in liquid nitrogen. Add 600 µl Buffer RL for every 20-30 mg of tissue (check for addition of β-mercaptoethanol before use), and 350 µl Buffer RL for tissue samples of less than 20 mg. Sample volume is not to exceed one-tenth of the Buffer RL volume.1b Cells in monolayer culture: Lysed or processed into cell suspension directly in culture flask, centrifuged to obtain cell precipitate, discarded the supernatant, added 600µl Buffer RL for every 6-10 cm2 of culture area, 350µl Buffer RL for less than 6cm2, and blown several times repeatedly to make the cells lysed sufficiently.1c Cell suspension: centrifuge at 12,000 rpm (~13,400 × g) for 1 min and discard the supernatant to obtain the cell precipitate. Add 600 µl Buffer RL for every 5×106-1×107 cells, and 350 µl Buffer RL for less than 5×106 cells, and blow several times repeatedly to fully lysate.Note: 1) Try to get rid of the cell culture medium, which may inhibit cell lysis affecting RNA yield.2) Try to keep the cells well suspended and well lysed, otherwise RNA yield is affected.2. After the sample is fully lysed, leave it at room temperature for 5 minutes to allow complete separation of the protein-nucleic acid complex.3. Centrifuge at 12,000 rpm for 2-5 min and remove the supernatant for the following operations.4. Add 1x volume (600µl or 350µl) of 70% ethanol (prepared without RNase water) to the solution obtained in step 3 and mix well.Note: The addition of ethanol may produce a precipitate that will not affect subsequent experiments.5. Add all of the solution obtained in the previous step to the Spin Columns RM in the collection tube. If you cannot add all of the solution to the column at once, transfer it in two passes, centrifuge at 12,000 rpm for 1 minute, and discard the waste solution. Place the column back into the collection tube.Note: The maximum loading capacity of the adsorption column is 100µg, do not overload as this will affect the yield and purity of the RNA.6. Add 350 µl Buffer RW1 to the adsorbent column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid and put the adsorbent column back into the collection tube.7. Preparation of DNase I mixture: Take 52 µl of RNase-Free Water, add 8 µl of 10×Reaction Buffer and 20 µl of DNase I (1 U/µl) to it, mix well, and prepare a final volume of 80 µl of reaction solution.8. Add 80µl of DNase I mixture directly to the adsorption column and incubate at 20-30°C for 15 minutes.9. Add 200 µl Buffer RW1 to the adsorbent column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid and put the adsorbent column back into the collection tube.10. Add 500µl Buffer RW2 to the column (check that anhydrous ethanol is added before use), centrifuge 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.11. Repeat step 10.12. 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 a few minutes to thoroughly dry the anhydrous ethanol in the adsorption column.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.).13. Transfer the adsorbent column into a new centrifuge tube, add 30-50 µl of RNase-Free Water to the middle of the adsorbent membrane, leave it at room temperature for 1 min, centrifuge at 12,000 rpm for 1 min, collect the RNA solution, and store the RNA at -70°C to prevent degradation.Note: 1) The volume of RNase-Free Wate should not be less than 30 µl, too small volume affects the recovery rate.2) If you want to increase the RNA yield, repeat step 13 with 30-50 µl of fresh RNase-Free Water.3) If the RNA concentration is to be increased, the resulting solution can be reintroduced into the adsorption column and step 13 repeated... Read More | Product contentS665868Component50 TStorageS665868ABuffer GL25 mLRTS665868BBuffer GW1 (concentrate)13 mLRTS665868CBuffer GW2 (concentrate)15 mLRTS665868DBuffer GE15 mLRTS665868EProteinase K2×1.25 mLRTS665868FSpin Columns DM with Collection Tubes50 setsRTProduct IntroductionThis kit is suitable Product contentS665868Component50 TStorageS665868ABuffer GL25 mLRTS665868BBuffer GW1 (concentrate)13 mLRTS665868CBuffer GW2 (concentrate)15 mLRTS665868DBuffer GE15 mLRTS665868EProteinase K2×1.25 mLRTS665868FSpin Columns DM with Collection Tubes50 setsRTProduct IntroductionThis kit is suitable for the extraction of genomic DNA from fresh saliva or saliva/preservation solution mixture.The purification process of this product does not require the use of toxic solvents such as phenol or chloroform, and ethanol precipitation is not necessary. The optimized buffer system enables DNA to bind heterogeneously to the silica matrix centrifugal adsorption column, and the inhibitors of PCR and other enzymatic reactions can be effectively removed by a two-step washing step, and finally eluted with a low-salt buffer or water to obtain high-purity DNA.The purified obtained can be directly used for enzyme digestion, PCR, Real-Time PCR, library construction, Southern Blot, molecular labeling and other downstream experiments.Self-contained reagent: anhydrous ethanolPre-experiment Preparation and Important Notes1. Repeated freezing and thawing of the sample should be avoided, as this may result in smaller fragments of extracted DNA and a decrease in the amount extracted.2. 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.3. Before use, please check whether Buffer GL appears to be crystallized or precipitated.Redissolve in a 56°C water bath.4. If the downstream experiments are sensitive to RNA contamination, 4 µL DNase-Free RNase A can be added in step 3(100 mg/mL).5. For prolonged storage of salivary DNA at room temperature, our Salivary DNA Preservation Tubes are recommended.Operation steps1. Add 400 µL of saliva sample or saliva/preservation solution mixture.Note: 1) Saliva mixtures added to the preservation solution require a 50°C water bath for 1 hour or an empty 50°C temperature chamber for 2 hours prior to extraction.2) If an increase in sample volume is required, multiply the volumes of Proteinase K, Buffer GL, and anhydrous ethanol in Steps 2-4, and the liquid can be transferred in multiple times in Step 5.2. Add 40 µL of Proteinase K.3. Add 400µL Buffer GL, vortex and shake to mix thoroughly, and water bath at 56℃ for 15-30 minutes.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, vortex for 15 seconds, and leave at room temperature for 2 minutes.4. Centrifuge briefly to remove water droplets from the inside of the tube cap. Add 400 µL of anhydrous ethanol and mix well by vortexing and shaking. Centrifuge briefly.Note: 1) Vortex and shake to mix immediately after adding Buffer GL and anhydrous ethanol.The addition of Buffer GL and anhydrous ethanol may produce a white precipitate that will not affect subsequent experiments.2) A sol-gel product may be formed after GL and anhydrous ethanol, in which case vigorous shaking or vortexing is recommended.3) The solution obtained in the previous step is added to the adsorption column in the Collection Tube.5. (Spin Column DM) in 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 (∼13,400 × g) for 1 min, pour off the waste solution in the collection tube, and put the adsorption column back into 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 put the adsorption 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 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 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 centrifuge tube (supplied), add 50-200 µL of Buffer GE or sterilized water to the middle of the adsorption column overhanging the column, let it stand at room temperature for 2-5 minutes, and centrifuge at 12,000 rpm for 1 minute to collect the DNA solution.-20°C to preserve DNA.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) Buffer GE preheated in a 65-70°C water bath and incubated at room temperature for 5 min before centrifugation can increase the yield.3) Because DNA preserved in water is subject to acidic hydrolysis, for long-term storage, elution with Buffer GE and storage at -20°C is recommended... Read More |