| Description | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | When apoptosis occurs, some DNA endonucleases will be activated. These endonucleases will cut off genomic DNA between nucleosomes and produce 180 bp-200 BP DNA fragments, which appear as a specific ladder pattern in agarose gel electrophoresis. When double strand or single strand breaks occurWhen apoptosis occurs, some DNA endonucleases will be activated. These endonucleases will cut off genomic DNA between nucleosomes and produce 180 bp-200 BP DNA fragments, which appear as a specific ladder pattern in agarose gel electrophoresis. When double strand or single strand breaks occur in genomic DNA, a large number of sticky 3'-oh ends will be generated, which can interact with YF under the catalysis of deoxyribonucleotide terminal transferase (TDT) ®/ CY dUTP binding can directly detect apoptotic cells by fluorescence microscopy or flow cytometry. This kind of method is called terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL). Because normal or proliferating cells have almost no DNA breaks, there is no 3'-oh formation and they can rarely be stained. TUNEL method can stain intact single apoptotic nuclei or apoptotic bodies in situ, can accurately reflect the typical biochemical and morphological characteristics of apoptosis, and can detect a very small number of apoptotic cells, so it is widely used in the study of apoptosis. This kit has a wide range of applications and can be used to detect apoptosis in frozen or paraffin sections, as well as cultured adherent cells or suspended cells. It can selectively detect apoptotic cells, but not necrotic cells or cells with DNA strand breaks caused by irradiation and drug treatment. This kit detects cell apoptosis with a short time-consuming, one-step staining reaction and can be detected after washing.Component： Instruction： Experimental materials (self provided)PBS buffer (1 x, pH~7.4). 0.2% Triton X -100 (PBS formulation). 0.1% Triton X -100 (PBS formulation, containing 5 mg/mLBSA)4% paraformaldehyde (prepared with PBS)Immunohistochemical penDewaxing solvent (paraffin section sample)Related reagents for paraffin section processingAnti fluorescence quenching and sealing agent. ddH2Oexperimental design. A. Positive control:Prepare positive control slides using DNaseI treatment. DNaseI can digest single or double stranded DNA and expose the 3 '- OH end, artificially causing cell apoptosis. One experiment per time is sufficient. (To verify if there are any issues with the experimental operation and reagent kit)B. Negative control:Use TUNEL Reaction Buffer without TdT Enzyme and replace TdT Enzyme with ddH2O. (Mainly to exclude non-specific staining caused by cell apoptosis, operational processes, and other reasons; and to adjust the exposure intensity of the shooting.)C. Experimental processing group.The experimental group operated normally according to the instructions.D. Experimental control group.The experimental group operated normally according to the instructions.Experimental steps1. Sample preparation:(1) For adherent cells or cell smearsa. Clean once with PBS.Note: If you are concerned that the cells on the cell smear may not adhere firmly, you can dry the sample to make the cells adhere more firmly.b. Fixation: Add an appropriate amount of 4% paraformaldehyde (prepared with PBS) and fix at 4 ℃ for 30 minutes. Clean twice with PBS.c. Translucency: Add an appropriate amount of 0.2% Triton X -100 (prepared with PBS) and let it penetrate at room temperature for 20 minutes. Clean twice with PBS.d. Step 2: TUNEL reaction.(2) For suspended cells or cell suspensionsa. Collect cells (3-5 x 106 cells), centrifuge at 1000 rpm for 5 minutes, and wash twice with PBS.b. Fixation: Add an appropriate amount of 4% paraformaldehyde (prepared with PBS) and resuspend the cells thoroughly. Fix at 4 ℃ for 30 minutes. Centrifuge at 2000 rpm for 5 minutes and clean twice with PBS.c. Translucency: Add an appropriate amount of 0.2% Triton X -100 (prepared with PBS) and let it penetrate at room temperature for 20 minutes. Centrifuge at 2000 rpm for 5 minutes and clean twice with PBS.d. Step 2: TUNEL reaction.(3) Paraffin tissue sectioninga. Dewaxing and hydration: Place the sliced samples sequentially in xylene I (10 min) → xylene II (10 min) → 100% ethanol I (5 min) → 100% ethanol II (5 min) → 95% ethanol (5 min) → 90% ethanol (5 min) → 80% ethanol (5 min) → 70% ethanol (5 min) → ddH2O rinse for 5 min, rinse twice.Note: Xylene is toxic and volatile. Please perform this operation in a fume hood.b. Use filter paper to dry the liquid around the sliced sample, and circle the sample contour with an immunohistochemical pen for downstream transparency and labeling.Note: If it is found that the contour circle of immunohistochemistry strokes is damaged in subsequent experimental operations, it needs to be redrawn in a timely manner.c. Transparency: Dilute 2 mg/mL of ProteinaseK solution with PBS in a ratio of 1:100 to a final concentration of 20 µ g/mL. Add 100 µ L dropwise to each sample to cover all sample areas. Incubate at 20-37 ℃ for 20 minutes.Note: Protein K can penetrate the cell membrane and nuclear membrane, allowing subsequent staining reagents to fully enter the nucleus for reaction and improve labeling efficiency. An excessively long incubation time increases the risk of tissue slices falling off the carrier film during subsequent washing steps, while a too short incubation time may result in insufficient permeability treatment and affect labeling efficiency. To obtain better results, the concentration, incubation time, and temperature of Protein K need to be optimized according to different types of tissue samples.d. Wash the slices twice with PBS, each time for 5 minutes. Use filter paper to remove excess liquid, and place the processed sample in a wet box to keep it moist.Note: Protein K must be washed thoroughly in this step, otherwise it will seriously interfere with subsequent labeling reactions.e. Step 2: TUNEL reaction.(4) Frozen tissue sectionsa. Fixation: Take out frozen sections and warm them back to room temperature. Add an appropriate amount of 4% paraformaldehyde (prepared with PBS) and fix at room temperature for 30 minutes. Wash twice with PBS for 10 minutes each time.Note: If you are concerned that formaldehyde cleaning may not be clean enough, it may affect the final dyeing effect. After formaldehyde fixation is completed, an appropriate amount of 2 mg/mL glycine can be added and washed for 10 minutes to neutralize the residual fixing solution, and then PBS cleaning can be carried out.b. Use filter paper to dry the liquid around the sliced sample, and circle the sample contour with an immunohistochemical pen for downstream transparency and labeling.Note: If it is found that the contour circle of immunohistochemistry strokes is damaged in subsequent experimental operations, it needs to be redrawn in a timely manner.c. Transparency: Dilute 2 mg/mL of ProteinaseK solution with PBS in a ratio of 1:100 to a final concentration of 20 µ g/mL. Add 100 µ L dropwise to each sample to cover all sample areas. Incubate at 20-37 ℃ for 20 minutes.Note: Protein K can penetrate the cell membrane and nuclear membrane, allowing subsequent staining reagents to fully enter the nucleus for reaction and improve labeling efficiency. An excessively long incubation time increases the risk of tissue slices falling off the carrier film during subsequent washing steps, while a too short incubation time may result in insufficient permeability treatment and affect labeling efficiency. To obtain better results, the concentration, incubation time, and temperature of Protein K need to be optimized according to different types of tissue samples.d. Wash the slices twice with PBS, each time for 5 minutes. Use filter paper to remove excess liquid, and place the processed sample in a wet box to keep it moist.Note: Protein K must be washed thoroughly in this step, otherwise it will seriously interfere with subsequent labeling reactions.e. Step 2: TUNEL reaction.(5) Positive treatment (only the positive control is subjected to this step, and other samples are directly subjected to the TUNEL reaction step)a. Dilute 10 x DNase I Buffer with ddH2O in a ratio of 1:10 to 1 x DNase I Buffer for later use.b. Drip 100 µ L of 1xDNase I Buffer onto the processed sample, covering all sample areas, and equilibrate at room temperature for 5 minutes.c. Dilute DNase I (2 U) with 1 x DNase I Buffer at a ratio of 1:100/ µ L) A working solution with a final concentration of 20 U/mL.d. Discard the buffer and add 100 µ Incubate DNase I working solution with a concentration of 20 U/mL at room temperature for 10 minutes.e. Discard DNase I working solution and clean twice with PBS.f. Step 2: TUNEL reaction.2. TUNEL reaction(1) Prepare TUNEL reaction solution (ready to use):/1 sample5sample10 sampleTdT enzyme1 µL5 µL10 µLYF®488/555/594/640 TUNEL Reaction Buffer49 µL245 µL490 µLTUNEL Total volume of reaction solution50 µL250 µL500 µL(2) For adherent cells, cell smears, or tissue sectionsa. Add 50 to each sample µ L TUNEL reaction solution, evenly cover the sample with the reaction solution. The appropriate time for dark incubation at 37 ℃ (recommended staining time for cells is 30 minutes to 1 hour, and tissue staining time is 2 hours).Note: 50 µ L TUNEL reaction solution is suitable for smear, slicing, or 96 well plates (other different well plates can adjust the volume of TUNEL reaction solution appropriately to cover cells). If the sample to be tested is a smear, slice, or in a 24 well plate, 12 well plate, or 6 well plate, anti evaporation film can be used, or self sealing bags or other appropriate materials can be used to cut circular plastic sheets slightly smaller than the holes. After adding TUNEL reaction solution dropwise, cover the sample to prevent the evaporation of TUNEL reaction solution and make the TUNEL reaction solution evenly cover the sample.b. Discard the TUNEL reaction solution, wash twice with PBS, and then wash three times with 0.1% Triton X -100 (PBS preparation, containing 5 mg/mL BSA) for 5 minutes each time. This way, free unreacted markers can be removed cleanly.c. (Optional) Add an appropriate concentration of 5 to each sample µ DAPI staining solution with a concentration of g/mL, incubated at room temperature in dark for 5 minutes. After staining, discard DAPI staining solution and wash twice with PBS for 5 minutes each time.d. (Optional) Slice sealing: Add 50 drops to each sample µ L anti fluorescence quenching sealing agent (anti fluorescence quenching sealing agent may not be suitable for certain dyes, it is recommended to conduct pre experimental testing for compatibility before the experiment), cover the cover glass, gently tap the cover glass with the blunt end of tweezers to remove bubbles and ensure complete sealing.e. Use filter paper to remove excess liquid and add 100 to the sample area µ Keep the sample moist with PBS and immediately observe under a fluorescence microscope.(3) For suspended cells or cell suspensionsa. Add 50 to each sample tube µ Gently resuspend cells in LTUNEL reaction solution and incubate at 37 ℃ in the dark for 30-1 hour. Gently resuspend cells with a micropipette every 15 minutes.b. Centrifuge at 2000 rpm for 5 minutes, discard TUNEL reaction solution, and wash twice with 0.1% Triton X -100 (PBS preparation, containing 5 mg/mLBSA) for 5 minutes each time. This way, free unreacted markers can be removed cleanly.c. Add 100 to each sample tube µ L concentration is 5 µ DAPI staining solution with a concentration of g/mL, incubated at room temperature in dark for 5 minutes.d. Join 400 µ L PBS resuspended cells and immediately detected with a flow cytometer or observed under a fluorescence microscope after smearing.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. when the staining background is heavy or non-specific staining is obvious, the staining time can be appropriately reduced. 3. it is recommended to add negative control and positive control groups during the experiment. 4. please wear mask and gloves when using component A. if it contacts the skin, please wash it with plenty of water immediately. 5. fluorescent dyes have quenching problems. Please try to avoid light to slow down fluorescence quenching. 6. for your safety and health, please wear experimental clothes and disposable gloves.Product parameters：490/515 nm；Scope of application：Late apoptosis detection, TUNEL Kit... Read More | Inquire | 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 contentY666144Component50 TStorageY666144ABuffer P115 mLRTY666144BBuffer P215 mLRTY666144CBuffer N320 mLRTY666144DBuffer PS15 mLRTY666144EBuffer PB10 mLRTY666144FBuffer PW (concentrate)10 mLRTY666144GBuffer EB10 mLRTY666144HGlass Beads2 gRTY666144IRNase A (10mg/mL)150 µLRTY666144JSpin Product contentY666144Component50 TStorageY666144ABuffer P115 mLRTY666144BBuffer P215 mLRTY666144CBuffer N320 mLRTY666144DBuffer PS15 mLRTY666144EBuffer PB10 mLRTY666144FBuffer PW (concentrate)10 mLRTY666144GBuffer EB10 mLRTY666144HGlass Beads2 gRTY666144IRNase A (10mg/mL)150 µLRTY666144JSpin Columns DM with Collection Tubes50 setsRTProductsThis kit is improved on the basis of common alkaline lysis method, the glass beads can effectively break the yeast cell wall, the new silica matrix membrane and buffer system can efficiently and specifically bind the plasmid DNA, and at the same time can maximize the removal of proteins and other impurities, the whole process is convenient and fast, no need to use toxic and harmful reagents, and can be processed at the same time for multiple samples. In addition to yeast cells, it can also be used in E. coli. Plasmid DNA extracted with this kit can be used in various molecular biology experiments, such as ligation, transformation, sequencing and library screening.Self-contained reagents: β-mercaptoethanol, 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 to Buffer P1, mix well, and store at 2-8℃.3. Anhydrous ethanol should be added to Buffer PW before first use according to the instructions on the reagent bottle label.4. Before use, please check whether Buffer P2 and Buffer N3 are crystallized or precipitated. If there is any crystallization or precipitation phenomenon, it can be clarified by taking a water bath at 37℃ for a few minutes to restore the clarity.5. Be careful not to touch Buffer P2 and Buffer N3 directly, and tighten the lid immediately after use.6. The amount of plasmid extracted is related to the yeast strain, plasmid copy number, culture conditions, etc. Usually, yeast plasmid copy number is very low, which is difficult to be detected by electrophoresis or spectrophotometer method.Procedure1. Take 1-5 ml of yeast culture (maximum 5×107 yeast cells, generally for Saccharomyces cerevisiae OD = 1.0, equivalent to 1-2×107 cells/ml) and add it to a centrifuge tube (self-provided), centrifuge for 30 seconds at 12,000 rpm (~13,400×g), collect the bacterial precipitate, and aspirate as much as possible to discard the supernatant.2. Add 250µl Buffer P1 to the bacterium (please check if RNase A has been added first) and resuspend the precipitate.3. Add 40mg of Glass Beads to the above mixture and vortex and shake for 10 minutes.4. Add 250 µl of Buffer P2 to the centrifuge tube, mix gently by turning up and down 6-8 times, and let stand at room temperature for 5-10 minutes, at which time the bacterial solution should become clear and viscous.Note: Mix gently, do not shake violently, so as not to interrupt the genomic DNA, resulting in genomic DNA fragments mixed in the extracted plasmid. If the solution does not become clear, it suggests that the amount of bacteria may be too large and the lysis is not complete, and the amount of bacteria should be reduced.5. Add 350 µl of Buffer N3 to the centrifuge tube and immediately mix gently up and down 6-8 times, at which point a white flocculent precipitate appears, and centrifuge at 12,000 rpm for 20 minutes.Note: Buffer N3 should be mixed immediately after addition to avoid localized precipitation.6. Column Equilibration: Add 200 µl of Buffer PS to the Spin Columns DM in the collection tube, centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid from the collection tube, and place the column back into the collection tube.7. Add the supernatant from step 5 to the adsorbent column that has been loaded into the collection tube, taking care not to aspirate the precipitate.Note: The maximum volume of the adsorption column is 750 µl, and the solution is passed through the column in 2 times.8. Centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube and place the adsorption column back into the collection tube.9. Add 150 µl Buffer PB to the adsorbent column, centrifuge at 12,000 rpm for 1 min, pour off the waste liquid in the collection tube, and put the adsorbent column back into the collection tube.10. Add 750 µl Buffer PW to the adsorption column (please check that anhydrous ethanol has been added first), centrifuge at 12,000 rpm for 1 minute, and pour off the waste liquid in the collection tube.11. Place the column back into the recovery collection tube and centrifuge at 12,000 rpm for 2 minutes, pouring off the waste liquid. Leave the 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.).12. Place the adsorbent column in a new centrifuge tube, add 50-100 µl of Buffer EB to the center of the adsorbent membrane dropwise, let it stand at room temperature for a few minutes, centrifuge at 13,000 rpm for 1 minute, and collect the plasmid solution into the centrifuge tube. Store the plasmid at -20°C.Attention:1) To increase the recovery efficiency of the plasmid, the resulting solution can be reintroduced into the adsorbent column, left at room temperature for a few minutes, centrifuged at 13,000 rpm for 1 minute, and the plasmid solution collected into a centrifuge tube.2) When the plasmid copy number is low or >10 kb, Buffer EB is preheated at 65-70°C in a water bath, which can increase the extraction efficiency.3) Usually yeast plasmids have very low copy number and are difficult to detect by electrophoresis or spectrophotometry. If the extracted plasmid is to be used in the next step of the experiment, it is usually recommended to use 1-5µl of the plasmid as PCR template, and 5-10µl of the plasmid for transformation of E. coli.4) Commercial high transformation efficiency receptor cells should be used for transformation of E. coli... Read More |