| Description | Firefly Luciferase Reporter Gene Assay Kit (Glow) is a glow-type quantitative detection kit characterized by high sensitivity and stable luminescent signals. It is suitable for high-throughput detection of firefly luciferase expression in mammalian cells. This product significantly enhances Firefly Luciferase Reporter Gene Assay Kit (Glow) is a glow-type quantitative detection kit characterized by high sensitivity and stable luminescent signals. It is suitable for high-throughput detection of firefly luciferase expression in mammalian cells. This product significantly enhances luminescent signal intensity, effectively improving detection sensitivity. It can be used to detect firefly luciferase reporter gene expression in ADCC Reporter Bioassays or cells. G1375755Component10 mL100 mLStorageG1375755AFirefly Luciferase Reaction Buffer (Glow)10 mL100 mL-20℃. Store in the dark.G1375755BFirefly Luciferase Substrate (Glow)1 vial1 vial-20℃. Store in the dark.he main features and differences among three One-Step Firefly Luciferase Reporter Gene Assay Kits from Aladdin are as follows:For high luminescent signal requirements and detection within 30 minutes: Recommended: Firefly Luciferase Reporter Gene Assay Kit (Glow Bright) (Cat. No. B1375760)For both luminescent signal and stability requirements, suitable for batch or continuous operation: Recommended: Firefly Luciferase Reporter Gene Assay Kit (Cat. No. F773892)For standard luminescent signal requirements: Recommended: Firefly Luciferase Reporter Gene Assay Kit (Glow) (Cat. No. G1375755)For routine laboratory detection, the Firefly Luciferase Reporter Gene Assay Kit (Glow Bright) (Cat. No. B1375760) is the recommended priority. Product Cat. No.Product NameLuminescence TypeSignal IntensitySensitivityHalf-LifePackagingSuitable for Batch SamplesOptimal Use After Mixing & Storing at -20°CF773892Firefly Luciferase Reporter Gene Assay KitGlowRelatively HighRelatively SensitiveUp to 2h1 bottle Buffer1 vial SubstrateVery Suitable1 MonthG1375755Firefly Luciferase Reporter Gene Assay Kit (Glow)GlowHighSensitive25-30min1 bottle Buffer1 vial SubstrateVery Suitable1 WeekB1375760Firefly Luciferase Reporter Gene Assay Kit (Glow Bright)GlowVery HighHighly Sensitive25-30min1 bottle Buffer1 vial SubstrateVery Suitable1 WeekInstructions for Use:1. User-Supplied MaterialsPBSMultichannel pipetteWhite or black opaque cell culture microplateLuminometer or microplate reader equipped with a luminescence detection module.2. Preparation Before Assay(1) Initial Reconstitution: Upon first use, transfer the entire contents of the Firefly Luciferase Reaction Buffer (Glow) bottle into the Firefly Luciferase Substrate (Glow) vial. Mix thoroughly until the substrate is completely dissolved. Aliquot the reconstituted detection reagent as needed. For long-term storage, aliquot and store at -70°C. For short-term storage, store aliquots at -20°C for no longer than one month. Use reconstituted reagent promptly.(2) Pre-assay: Before each experiment, equilibrate the aliquoted, frozen detection reagent to room temperature.3. Procedure(1) Equilibrate Plates: Remove the cell culture plate from the incubator and allow it to stand at room temperature for 5-15 minutes.Note: Use white or black opaque cell culture plates to minimize signal interference between wells.(2) Add Detection Reagent: Using a multichannel pipette, add the equilibrated Firefly Luciferase Reaction Buffer (Glow) containing the substrate to each cell culture well. The volume added should be equal to the volume of the culture medium in the well. For example:* 96-well plate: Typically add 80-100 µL culture medium; add 80-100 µL detection reagent.* 384-well plate: Typically add 20-30 µL culture medium; add 20-30 µL detection reagent.(3) Mix to Lyse Cells: To ensure complete cell lysis, place the plate on an orbital shaker or an instrument with shaking capability. Shake at medium to high speed at room temperature for 5 minutes.Note: A shaking time of 5 minutes is recommended. This can be adjusted based on cell density to ensure complete lysis and stable luminescent results.(4) Detection: Following the shaking step, immediately measure firefly luciferase reporter gene activity using a luminometer or microplate reader.Note: For optimal results, perform the detection as soon as possible after adding the detection reagent.PrecautionsInstrument Selection: Any instrument capable of detecting chemiluminescence is suitable for use with this kit. However, background signal levels and measured values for identical samples may vary between different detectors. Values obtained from different instruments for the same sample are not directly comparable. To prevent well-to-well interference, the use of opaque white or black cell culture plates is strongly recommended.Consistent Conditions: Luminescent signals can be influenced by environmental factors such as medium components and temperature. Ensure consistent detection conditions across all samples within an experiment.Temperature Sensitivity: The enzymatic reaction is temperature-sensitive. It is essential to equilibrate both the detection reagent and the cell culture plate to room temperature before adding the reagent and performing the assay.Reagent Stability: To maintain the stability of the luciferase assay reagent, aliquot it appropriately after reconstitution. For long-term storage, store aliquots at -70°C protected from light. For short-term storage, store at -20°C for no longer than one month. Use aliquots promptly and avoid repeated freeze-thaw cycles.Multiple Plate Assay: If detecting multiple cell culture plates simultaneously, strive to maintain consistent incubation time after adding the detection reagent to each plate before reading. This ensures optimal and comparable results across plates.Emission Wavelength: The bioluminescence catalyzed by firefly luciferase exhibits a peak emission wavelength of 560 nm... Read More | This reagent kit is suitable for simultaneously isolating and purifying genomic DNA, total RNA, and total protein from the same cell or tissue sample. This product does not require dividing the sample into three parts to extract DNA, RNA, and protein separately, nor does it require dividing the This reagent kit is suitable for simultaneously isolating and purifying genomic DNA, total RNA, and total protein from the same cell or tissue sample. This product does not require dividing the sample into three parts to extract DNA, RNA, and protein separately, nor does it require dividing the purified total nucleic acid into two parts before purifying DNA and RNA separately. Therefore, it can maximize the recovery of DNA, RNA, and protein, and can be used for the purification of nucleic acid and protein in small and rare samples. The purified DNA, RNA, and protein can be eluted separately and directly applied to various downstream molecular biology operations. This reagent kit does not contain toxic substances such as phenol and chloroform, and does not require ethanol precipitation. The operation is simple and fast. The extracted genomic DNA can be used for PCR, Real time PCR, SouthBlot, Dot Blot, comparative genomic hybridization (CGH), gene analysis, and SNP analysis; Total RNA can be applied in experiments such as RT-PCR, cDNA synthesis, Northern Blot, Dot Blot, and gene chips; Total protein can be applied in electrophoresis and Western Blot, among others. A665492 Component 50 T Storage A665492A Buffer RL 35 mL RT A665492B Buffer RW1 40 mL RT A665492C Buffer RW2 (concentrate) 11 mL RT A665492D RNase-Free Water 10 mL RT A665492E Buffer GW1 (concentrate) 13 mL RT A665492F Buffer GW2 (concentrate) 15 mL RT A665492G Buffer GE 15 mL RT A665492H Buffer PZ 60 mL RT A665492I Buffer PLS 15 mL RT A665492J Spin Columns DM with Collection Tubes 50 sets RT A665492K Spin Columns RM with Collection Tubes 50 sets RT A665492L Collection Tubes 100 EA RT A665492M RNase-Free Centrifuge Tubes (1.5 mL) 100 EA RTSelf prepared reagents:β- Mercaptoethanol (for newly opened or RNA extraction), 70% ethanol (prepared with water without RNase), and anhydrous ethanol.Preparation and important precautions before the experiment:To prevent RNase pollution, attention should be paid to the following aspects:1) Use plastic products and gun heads without RNase to avoid cross contamination.2) Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use, while plastic containers can be soaked in 0.5 M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.3) The solution should be prepared 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 freeze-thaw cycles, otherwise it will affect the quality of DNA, RNA, and protein extraction. The sample can be stored in Buffer RL at -70 ℃ for one month.3. Please add Buffer RL before use β- Mercaptoethanol, 1 ml Buffer RL with 10 µ L β- Mercaptoethanol. join β- The buffer RL room temperature of mercaptoethanol can be stored for one month.Before the first use, anhydrous ethanol should be added to Buffer RW2, Buffer GW1, and Buffer GW2 according to the instructions on the reagent bottle label.5. Before use, please check if there is any crystallization or precipitation in the Buffer RL. If there is any crystallization or precipitation, please dissolve it again in a 56 ℃ water bath.6. All centrifugation steps are performed using a desktop centrifuge at room temperature. Operation steps:1. Material processing1a The cells cultured on the wall should be first processed into cell suspension (maximum extraction amount of 107 cells), collected cells, discarded the culture medium, and added 600 cells µ L Buffer RL (check if it has been added before use) β- Mercaptoethanol), repeatedly blow and beat to fully decompose.Attention: It is necessary to discard the culture medium completely, otherwise it will affect the lysis and subsequent nucleic acid purification steps.1b Take no more than 30 mg of animal tissue, grind it into fine powder with liquid nitrogen, and add 600 µ Buffer RL (check if it has been added before use) β- Mercaptoethanol, or directly add 600 µ L Buffer RL (check if it has been added before use) β- Mercaptoethanol, homogenization treatment.Attention: The homogenate should be sufficient, otherwise it will affect RNA production.2. Centrifuge the solution obtained in the previous step at 12000 rpm (~13400 × g) for 3-5 minutes. Carefully add the supernatant to the spin columns DM that have been loaded into the collection tube. Centrifuge at 12000 rpm for 30-60 seconds and collect the filtrate. Place the adsorption column DM in a new 2 ml collection tube at room temperature or 4 ℃ for DNA extraction. Attention: Ensure that there is no liquid residue on the adsorption column, and if necessary, repeat centrifugation until all liquids pass through the membrane of the adsorption column. Total RNA extraction3. Add 1 volume of 70% ethanol (prepared without RNase water) to the filtrate obtained in step 2, and mix well.4. 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 completely at once, it can be transferred in stages. Centrifuge at 12000 rpm for 20 seconds and retain the liquid in the collection tube for protein extraction.5. Place the adsorption column RM into a new 2ml collection tube and add 700 to the adsorption column RM µ L Buffer RW1, centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column RM into the recovery manifold.6. Add 500 to the adsorption column RM µ Buffer RW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column RM back into the 2 ml collection tube.7. Repeat step 6.Centrifuge at 8.12000 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. Attention: 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.).9. Place the adsorption column RM in a new 1.5 ml centrifuge tube without RNase, and add 30-50 to the middle of the adsorption column RM µ Place RNase Free Water at room temperature for 2-5 minutes, 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 9 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 9.Genomic DNA extraction10. Add 500 to the adsorption column DM µ Buffer GW1 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column DM into the recovery tube.11. Add 500 to the adsorption column DM µ Buffer GW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 2 minutes, discard the waste liquid in the collection tube, and place the adsorption column DM into the recovery tube. Attention: To further improve DNA purity, repeat step 11.Centrifuge at 12.12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column DM at room temperature for a few minutes to thoroughly dry the ethanol in the column. Attention: 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 DM in a new centrifuge tube and add 100 to the middle of the adsorption column DM by suspending it in the air µ L Buffer GE, leave at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 2 minutes, collect DNA solution, and store DNA at -20 ℃.Attention:1) The volume of Buffer GE should not be less than 100 µ l. Small volume affects the recovery rate.2) If we want to increase DNA production, we will µ Add a new Buffer GE to the adsorption column and repeat step 13; If you want to increase the DNA concentration, you can add the DNA eluent obtained in step 13 back onto the adsorption column and repeat step 13.Protein extraction14. Add 1 volume of Buffer PZ to the RNA extraction effluent (i.e. the solution obtained in step 4), mix well, and let it stand at room temperature for 10-30 minutes.Centrifuge at 15.12000 rpm for 10 minutes and discard the supernatant.16. Add 500 µ Centrifuge at 12000 rpm for 1 minute with 70% ethanol, and try to absorb the supernatant as much as possible.17. Place the centrifuge tube at room temperature for a few minutes to dry the precipitate.Attention: The purpose of this step is to remove residual ethanol. Excessive drying can make protein precipitation difficult to dissolve, and incomplete drying of residual ethanol can affect protein loading.18. Add 100 µ L Buffer PLS to obtain protein solution.Attention:1) The protein samples obtained by dissolving with Buffer PLS are suitable for SDS-PAGE and Western Blot detection, but not for Bradford method for protein quantification. If Bradford method is needed for protein quantification, 5% SDS can be used to dissolve the protein, or suitable protein dissolution buffer can be selected based on downstream experiments.2) The amount of dissolved protein buffer added is determined based on the initial sample size and specific downstream test requirements.3) The dissolved protein can be stored at -20 ℃ for several months and at 2-8 ℃ for several days.If protein samples require SDS-PAGE electrophoresis, the following operations can be performed:19. Add protein loading buffer to the protein sample, denature at 95 ℃ for 5-10 minutes, and cool the sample to room temperature. Centrifuge at 20.12000 rpm for 1 minute, extract the supernatant for downstream SDS-PAGE or Western blot tests... Read More | Products contentN665993Component240 TStorageN665993AIndex N501 Primers for Illumina240 µL-20℃. Avoid freeze/ Thaw cycle.N665993BIndex N925-N948 Primers for Illumina24×10 µL-20℃. Avoid freeze/ Thaw cycle. Products Introduction This kit is a companion kit to the transposase-Products contentN665993Component240 TStorageN665993AIndex N501 Primers for Illumina240 µL-20℃. Avoid freeze/ Thaw cycle.N665993BIndex N925-N948 Primers for Illumina24×10 µL-20℃. Avoid freeze/ Thaw cycle. Products Introduction This kit is a companion kit to the transposase-based Rapid DNA Library Construction Kit for Illumina platform library construction. Each kit contains one N5 primer and 24 N7 primers, which can be used to prepare 24 different single-ended Index libraries. All reagents provided in the kits have been subjected to stringent quality control and functional validation to maximize the stability and reproducibility of library construction. The libraries can be used for sequencing on Illumina platforms such as HiSeq X-10/4000/2500/2000 and MiSeq. Provide your own instruments, reagents and consumables1. Magnetic frame: DynaMagTM-2 is recommended.2. DNA purification and recovery kit: It is recommended to use Kangwei DNA purification and recovery kit by magnetic bead method.3. DNA building kit: It is recommended to use the Kangwei Century transposase method second-generation sequencing rapid DNA building kit.4. Anhydrous ethanol.5. Reaction tubes: It is recommended to use low adsorption PCR tubes with 1.5 ml centrifuge tubes;Tip: It is recommended to use a high quality filter tip to prevent contamination of kits and library samples. Pre-experiment Preparation and Important NotesPlease centrifuge briefly before opening the cap so that the liquid collects at the bottom of the tube to avoid cross-contamination between different primers. ProcedureFor the use of the CombiVision Second Generation Sequencing Multisample Primer Kit, please follow the CombiVision Second Generation Sequencing Rapid DNA Library Kit protocol.Index N501 Primer for IlluminaIndex N901-N996 Primer for Illumina... Read More | O665690 Component 50T Storage O665690A DNase I 1000 U -20℃.Avoid freeze/thaw cycle. O665690B 10×Reaction Buffer 1000 µL -20℃.Avoid freeze/thaw cycle. O665690C Buffer RLS 40 mL RT O665690D Buffer RW1 40 mL RT O665690E Buffer RW2 (concentrate) 11 mL RT O665690F RNase-Free Water O665690 Component 50T Storage O665690A DNase I 1000 U -20℃.Avoid freeze/thaw cycle. O665690B 10×Reaction Buffer 1000 µL -20℃.Avoid freeze/thaw cycle. O665690C Buffer RLS 40 mL RT O665690D Buffer RW1 40 mL RT O665690E Buffer RW2 (concentrate) 11 mL RT O665690F RNase-Free Water 10 mL RT O665690G Spin Columns FS with Collection Tubes 50 EA RT O665690H Spin Columns RM with Collection Tubes 50 EA RT O665690I RNase-Free Centrifuge Tubes (1.5 mL) 50 EA RTProduct IntroductionThis kit is suitable for extracting RNA from a wide range of plants, even from plants rich in polysaccharides and polyphenols, high quality RNA can be successfully extracted, such as rice leaves, wheat leaves, corn leaves, tobacco leaves, pine needles, ginkgo leaves, poplar leaves, pomegranate leaves, holly leaves, apples, peaches, pears, tomatoes, cherries, apricots, bananas, grapes, loquats, cinnamon rinds, cinnamon pulp, lychee fruit rinds, lychee pulp, soybean, peanut, corn, potato tuber, moonflower petal, pomegranate petal, shiitake mushroom, flat mushroom and other samples. The unique lysate formula can rapidly inactivate the RNA enzyme in the cell, effectively remove the effect of polysaccharide and polyphenol on RNA extraction, without the need for phenol, chloroform and other reagents, while using silicon matrix membrane adsorption of RNA for purification, the total RNA extracted is highly pure, without the contamination of genomes, proteins and other impurities, and can be used for Real Time RT-PCR, RT-PCR, It can be used for Real Time RT-PCR, RT-PCR, Northern Blot, Dot Blot, in vitro translation and other downstream experiments.RNA yieldSelf-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.(2) 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 rate and quality of RNA extraction.3. If Buffer RLS produces a precipitate, heat to dissolve it and leave at room temperature.4. Please add β-mercaptoethanol to Buffer RLS before use, add 20µl β-mercaptoethanol to 1ml Buffer RLS. Buffer RLS with β-mercaptoethanol can be stored for 1 month at room temperature.5. Anhydrous ethanol should be added according to the instructions on the reagent bottle label before using Buffer RW2 for the first time. Operation steps1. Homogenization: Take 50-100mg of plant tissue and quickly grind it into powder in liquid nitrogen, add 500µl of Buffer RLS (please check whether β-mercaptoethanol is added before use), and immediately mix it by vortexing with vigorous shaking.Note: For materials that are extremely rich in water content, such as watermelon pulp, tomato, pear pulp, etc., more material can be added appropriately, up to 200 mg; for starch-rich samples or mature leaves, the amount of Buffer RLS can be increased appropriately, up to 700 µl.2. Centrifuge at 12,000 rpm (~13,400 x g) for 2 min at 4°C.3. Transfer the supernatant into the filter columns (Spin Columns FS) that have been loaded into the collection tubes, centrifuge at 12,000 rpm at 4°C for 1 minute, carefully aspirate the supernatant in the collection tubes and transfer it to new RNase-Free centrifugation tubes (self-provided), avoiding the tip of the gun from touching the cell debris precipitation in the collection tubes as much as possible.4. Slowly add 0.5 times the volume of the supernatant in anhydrous ethanol, mix well (a precipitate may appear), and transfer the resulting solution together with the precipitate to a Spin Columns RM in a collection tube, or in two batches if you cannot add all of the solution at once. centrifuge the column for 1 minute at 12,000 rpm at 4°C. Dispose of the spent solution and place the column back into the collection tube. Centrifuge at 12,000 rpm for 1 minute at 4°C, discard the spent solution and return the column to the collection tube.5. Add 350 µl of Buffer RW1 to the adsorbent column RM, centrifuge at 12,000 rpm at 4°C for 1 min, discard the waste solution and put the adsorbent column back into the collection tube.6. Preparation of DNase I mixture: Take 52µl of RNase-Free Water, add 8µl of 10×Reaction Buffer and 20µl of DNase I (1U/µl) to it, mix well, and prepare a final volume of 80µl of reaction solution.7. Add 80µl of DNase I mixture directly to the adsorption column and incubate at 20-30°C for 15 minutes.8. Add 350 µl of Buffer RW1 to the adsorbent column RM, centrifuge at 12,000 rpm at 4°C for 1 min, discard the waste solution and put the adsorbent column back into the collection tube.9. Add 500 µl of Buffer RW2 to the adsorbent column RM (check that anhydrous ethanol is added before use), centrifuge at 12,000 rpm for 1 minute at 4°C, discard the waste solution and put the adsorbent column back into the collection tube.10. Repeat step 9.11. Centrifuge at 12,000 rpm for 2 minutes at 4°C.Note: The purpose of this step is to remove residual ethanol from the adsorption column; ethanol residue can interfere with subsequent enzymatic reactions (zymography, PCR, etc.).12. Load the adsorption column RM into new RNase-Free Centrifuge Tubes (1.5 ml), add 30-50 µl of RNase-Free Water dropwise to the middle part of the adsorption membrane overhang, leave it at room temperature for 2 min, and centrifuge at 12,000 rpm at 4°C for 1 min, and store the resulting RNA solution at -70°C to prevent degradation.Note: 1) The volume of RNase-Free Water 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 12 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 12 repeated... 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 |