计算溶液所需的质量、体积或浓度。
为什么选择此级别
，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
室温。常规运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
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产品简介
本试剂盒是进行微生物组分析的专用样品制备解决方案，适用于从拭子、血液、痰液、肺泡灌洗液等混合样本中纯化和富集细菌、真菌等病原微生物DNA/RNA。用本试剂盒纯化的微生物DNA/RNA适用于多种下游应用，包括全基因组测序分析、基于16S rDNA的高灵敏度微生物组分析、以及宏基因组鸟枪法测序分析
自备试剂及耗材
1. 带有气溶胶屏障的无菌移液器吸头，可防止交叉污染
2. 微量离心管（2 mL/1.5 mL）
3. PBS缓冲液（仅某些样品需要）
4. 异丙醇
实验前准备及重要注意事项
1. 本试剂盒旨在从完整的微生物细胞中分离DNA/RNA，为了保证微生物DNA/RNA的最佳回收效率，样品应保证新鲜。
2. 为避免由于污染造成的错误结果，请保持工作区域清洁和穿防护服，并合理设置对照品进行质控。采用适当措施处理样品材料，降低交叉污染的风险。
3, Buffer GW1（concentrate）和Buffer GW2（concentrate）根据瓶身信息加入相应量的乙醇。
操作步骤
1. 样本前处理：
1a：尿液、胸腹水、脑脊液等非粘稠体液样本
直接取400 μL样本，进行第2步操作。
1b：拭子类样本（如鼻、咽及肛拭子等）
涡旋振荡混匀后，直接取400 μL进行第2步实验。
1c：痰液、肺泡灌洗液样本
取适量液化痰液样本至1.5 mL 离心管中（液化方法推荐使用1.5倍体积的BufferGB1，本试剂盒不提供），12,000 rpm离心5 min，弃上清，使用400μLPBS重悬沉淀后提取。对于含有少量粘稠痰液的肺泡灌洗液，将尽量多的肺泡灌洗液样本先进行离心，小心去除上清，留取下层粘稠部分（含痰液、细胞、菌体）参照痰液样本进行液化处理。
1d：血液类样本
血清、血浆及少量全血样本（少于200 μL）可直接取400 μL（少量全血使用PBS补足）进行第2步实验，大体积全血样本推荐使用红细胞裂解液（CW0613）处理后再进行提取。
2. 向 Lysis Tube 中加入400 µL 样本、20 μL Proteinase K和400 µL Buffer SL-A，于恒温混匀仪上以65℃最大振速（2500~2900 rpm）处理10 min。短暂离心后加入400 µL Buffer GL，继续65℃最大振速（2500~2900 rpm）震荡处理10 min。
3. 室温12000 rpm离心1min，小心吸取全部上清至新离心管中，并加入400 µL异丙醇，涡旋混匀，瞬时离心使溶液收集至管底。
4. 将步骤3所得溶液（包括形成的沉淀）全部加入到已装入收集管的吸附柱，若一次不能加完溶液，可分多次转入（每次不超过700µL）。12000 rpm离心1分钟，倒掉收集管中的废液，将吸附柱重新放回收集管中。
5. 向吸附柱中加入500 μL Buffer GW1（使用前检查是否已加无水乙醇），12000 rpm 离心1分钟，倒掉收集管中的废液，将吸附柱重新放回收集管中。重复此步骤一次。
6. 向吸附柱中加入500 μL Buffer GW2（使用前检查是否已加无水乙醇），12000 rpm 离心1分钟，倒掉收集管中的废液，将吸附柱重新放回收集管中。重复此步骤一次。
7. 12,000 rpm离心2分钟，倒掉收集管中的废液。将吸附柱置于室温数分钟，彻底晾干（5~10min）。
8. 将吸附柱置于一个新离心管（自备）中，向吸附柱中间部位悬空加入70 μL RNase-Free Water，室温放置5分钟，12000 rpm离心1分钟，收集核酸溶液，-20℃保存。
Products content
Products Introduction
This 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 consumables
Sterile 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 Notes
1. 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.
procedure
1. 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℃.
规格
英文名称
Pathogenic Microbiome DNA/RNA Kit
储存与运输
储存条件
室温
运输条件
常规运输
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
C of A & Other Certificates(BSE/TSE, COO):
输入批号以搜索分析图谱:
技术文档和文章
溶液计算器
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