| 货号 (SKU) | 包装规格 | 是否现货 | 价格 | 数量 |
|---|---|---|---|---|
| U665923-50T |
50T |
期货  |
| |
| U665923-200T |
200T |
期货 |
|
| 英文名称 | Universal Genomic DNA Kit | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| 储存温度 | 室温 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 运输条件 | 常规运输 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 产品介绍 |
产品内容：
产品简介： 本试剂盒适合于从新鲜或冷冻的动物组织、细胞、血液、细菌等多种样品中提取 高纯度总DNA。本品可纯化获得分子量最大为50 kb的DNA片段，纯化过程不需使用苯 酚或氯仿等有毒溶剂，无需乙醇沉淀。本试剂盒采用优化的缓冲体系使裂解液中的 DNA高效特异的结合到硅基质离心吸附柱上， PCR和其他酶促反应的抑制剂可通过两 步洗涤步骤被有效去除，最后使用低盐缓冲液或水洗脱，即可得到高纯度DNA。纯化 得到的DNA可以直接用于酶切、 PCR、Real-Time PCR、文库构建、 Southern Blot、 分子标记等下游实验。 自备试剂:无水乙醇 Enzymatic Lysis Buffer（提取革兰氏阳性菌基因组DNA时须准备）。 自备试剂： Enzymatic Lysis Buffe配方： 20 mM Tris ，pH8.0；2 mM Na2-EDTA；1.2% Triton自备试剂： X-100；终浓度为20 mg/mL的Lysozyme（溶菌酶）。 实验前准备及重要注意事项： 1. 样品应避免反复冻融，否则会导致提取的DNA片段较小且提取量下降。 2. 如果提取次生代谢产物大量积累或细胞壁厚的细菌培养物的基因组，建议在对数 生长期早期收集样品。 3. 第一次使用前应按试剂瓶标签的说明在Buffer GW1和Buffer GW2中加入无水乙醇。 4. 使用前请检查Buffer GTL和Buffer GL是否出现结晶或者沉淀，如有结晶或者沉淀， 请将Buffer GL和Buffer GTL于56℃水浴重新溶解。 5. 如果下游实验对RNA污染比较敏感，可以在加入Buffer GL前加入4 μL DNase-Free 的RNase A（100 mg/mL）， RNase A 本试剂盒并未提供。 操作步骤： i 血液及细胞样本基因组提取 1. 材料处理 1a. 如果提取材料为哺乳动物抗凝血液（无核红细胞），可直接向50-200μL新鲜或冷 冻的抗凝血液样品中加入Buffer GTL补足至200μL； 1b. 如果提取材料为禽类、鸟类、两栖类或更低级生物的抗凝血液，其红细胞为有核 细胞，取5-10 μL新鲜或冷冻的抗凝血液样品，加入Buffer GTL补足至200 μL； 1c. 贴壁培养的细胞应先处理为细胞悬液（最大提取量为5×10⁶个细胞）， 2,000rpm （400×g）离心5分钟，弃尽上清，加200 μL GTL，振荡至样品彻底悬浮； 注意：如需去除RNA，可在上述步骤完成后，加入4 μL浓度为100 mg/mL的RNase A溶液，涡旋15秒,室温放置2分钟。 2. 加入20 μL Proteinase K。 3. 加入200 μL Buffer GL，涡旋震荡充分混匀， 56℃水浴10分钟。 4. 短暂离心以去除管盖内壁的水珠。加入200μL无水乙醇，涡旋震荡充分混匀。短暂离心。 注意：1）加入Buffer GL和无水乙醇后要立即涡旋震荡混匀。 2）加入Buffer GL和无水乙醇后可能会产生白色沉淀，不会影响后续实验。一些组织在加入Buffer GL 和无水乙醇后可能形成溶胶状产物，此时推荐进行剧烈震荡或涡旋处理。 5. 上一个步骤中所得溶液全部加入到已装入收集管的吸附柱（Spin Columns DM）中，若一次不能加完溶液，可分多次转入。 12,000 rpm（～13,400 ×g） 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。 6. 向吸附柱中加入500 μL Buffer GW1 （使用前检查是否已加入无水乙醇），12,000 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。 7. 向吸附柱中加入500 μL Buffer GW2（使用前检查是否已加入无水乙醇），12,000 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。 注意：如需进一步提高DNA纯度，可重复步骤7。 8. 12,000 rpm离心2分钟，倒掉收集管中废液。将吸附柱置于室温数分钟，以彻底晾干。 注意：这一步的目的是将吸附柱中残余的乙醇去除，乙醇的残留会影响后续的酶促反应（酶切、PCR等）。 9. 将吸附柱置于一个新的离心管（自备）中，向吸附柱的中间部位悬空加入50-200μL Buffer GE或灭菌水，室温放置2-5分钟，12,000 rpm离心1分钟，收集DNA溶液，-20℃保存DNA。 注意： 1）如果下游实验对pH值或EDTA敏感，可以用灭菌水洗脱。洗脱液的pH值对洗脱效率有很 大影响，若用水做洗脱液应保证其pH值在7.0-8.5（可以用NaOH将水的pH值调到此范围），pH值 低于7.0时洗脱效率不高。 2） Buffer GE在65-70℃水浴预热，离心之前室温孵育5分钟可以增加产量；用另外的50-200μL Buffer GE或 灭菌水再次洗脱可以增加产量。 3）如果要提高DNA的终浓度，可将得到的溶液重新加入到吸附柱中，室温放置2-5分钟，12,000 rpm离心1分钟；若洗脱体积小于200 μL，可以增加DNA的终浓度，但可能会减少总产量。如果DNA 的量小于1 μg，推荐用50 μL Buffer GE或灭菌水洗脱。 4）因为保存在水中的DNA会受到酸性水解作用的影响，如需长期保存，推荐用Buffer GE洗脱并于-20℃保存。 ii 动物组织基因组提取 1. 材料处理 如果提取材料为动物组织，取25 mg（脾组织用量应少于10 mg）；如果材料为鼠 尾，取一段长度为0.4-0.6 cm的大鼠鼠尾或两段长度为0.4-0.6 cm的小鼠鼠尾。 1a.样本进行液氮研磨或切成小块后置于1.5 mL离心管中，加入180 μL Buffer GTL， 将不同样品做好标记。 1b. 若使用匀浆器处理样本，匀浆前向样本中加入不超过80μL Buffer GTL，匀浆后 加入100 μL Buffer GTL。 注意： 1）确保各组织的量不要超出推荐范围。 2）组织样本在加入Buffer GTL之前用液氮研磨或加入Buffer GTL用匀浆器匀浆处理，可以增加裂解效率。 2.加入20 μL Proteinase K，涡旋震荡使样品彻底混匀。 56℃水浴，直至组织完全裂 解，孵育过程中可每隔一段时间颠倒或震荡离心管使样品分散。 注意： 1）不同组织消化时间不同，通常1-3小时即可完成，鼠尾需要消化6-8小时，必要时过夜消 化，不会影响后续操作。 2）如果孵育和涡旋震荡后仍然有胶状物质，延长56℃孵育时间或再加入20 μL Proteinase K消化。 3）如需去除RNA，可在上述步骤完成后，加入4μL的浓度为100 mg/mL的RNase A溶液，涡旋15秒，室温放置5-10分钟。 3. 加入200 μL Buffer GL，涡旋震荡充分混匀， 70℃水浴10分钟。短暂离心后加入200 μL无水乙醇，涡旋震荡充分混匀。 注意： 1）加入Buffer GL和无水乙醇后要立即涡旋震荡混匀。 2） 加入Buffer GL和无水乙醇后可能会产生白色沉淀，不会影响后续实验。 一些组织（如脾，肺）在 加入Buffer GL和无水乙醇后可能形成溶胶状产物，此时推荐进行剧烈震荡或涡旋处理。 4. 短暂离心，将步骤3所得溶液全部加入到已装入收集管的吸附柱（Spin Columns DM）中，若一次不能加完溶液，可分多次转入。 12,000 rpm（～13,400×g ）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入无水乙醇）， 12,000 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。 6.向吸附柱中加入500 μL Buffer GW2（使用前检查是否已加入无水乙醇）， 12,000 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。 注意：如需进一步提高DNA纯度，可重复步骤6。 7. 12,000 rpm离心2分钟，倒掉收集管中废液。将吸附柱置于室温数分钟，以彻底晾干。 注意：这一步的目的是将吸附柱中残余的乙醇去除，乙醇的残留会影响后续的酶促反应（酶切、PCR等）。 8. 将吸附柱置于一个新的离心管（自备）中，向吸附柱的中间部位悬空加入50-200μL Buffer GE或灭菌水，室温放置2-5分钟，12,000 rpm离心1分钟,收集DNA溶液， -20℃ 保存DNA。 注意： 1）如果下游实验对pH值或EDTA敏感，可以用灭菌水洗脱。洗脱液的pH值对洗脱效率有很 大影响，若用水做洗脱液应保证其pH值在7.0-8.5（可以用NaOH将水的pH值调到此范围），pH值 低于7.0时洗脱效率不高。 2）Buffer GE在65-70℃水浴预热，离心之前室温孵育5分钟可以增加产量；用另外的50-200 μL Buffer GE或灭菌水再次洗脱可以增加产量。 3）如果要提高DNA的终浓度，可将得到的溶液重新加入到吸附柱中，室温放置2-5分钟，12,000 rpm离心1分钟；若洗脱体积小于200μL，可以增加DNA的终浓度，但可能会减少总产量。如果 DNA的量小于1 μg，推荐用50 μL Buffer GE或灭菌水洗脱。 4）因为保存在水中的DNA会受到酸性水解作用的影响，如需长期保存，推荐用Buffer GE洗脱并于 -20℃保存。 iii 血液及细胞样本基因组提取 1. 细菌样本预处理 1a. 革兰氏阴性菌 （1）取细菌培养物1-5mL（10⁶-10⁸个细胞，最多不超过2×10⁹个细胞）置于离心 管（自备）中， 12,000 rpm（~13,400 ×g）离心1分钟，尽量吸净上清。 （2）向沉淀中加入180 μL Buffer GTL，振荡使菌体重悬。 （3）加入20 μL Proteinase K，涡旋混匀， 56℃孵育，直至菌体完全裂解，孵育 过程中每隔一段时间颠倒或震荡离心管使样本分散。 注意：如需去除RNA，可在上述步骤完成后，加入4 μL浓度为100 mg/mL的RNase A溶液，震荡混匀，室温放置5-10分钟。 （4）加入200 μL Buffer GL，涡旋震荡混匀。 1b. 革兰氏阳性菌 （1）取细菌培养物1-5 mL（10⁶-10⁸个细胞，最多不超过2×10⁹个细胞）置于离心 管（自备）中， 12,000 rpm离心1分钟，尽量吸净上清。 （2）加入180 μL Enzymatic Lysis Buffer（自备）使菌体重悬。 （3）37℃孵育30分钟。 （4）加入20 μL Proteinase K涡旋震荡，充分混匀。加入200 μL Buffer GL，涡旋震荡混匀。 56℃孵育30分钟。 注意： 1）如果需要，95°C孵育15分钟可以使病原体失活，但是95°C孵育会造成一些DNA的降解。 2）如需去除RNA，可在上述步骤完成后，加入4μL浓度为100 mg/mL的RNase A溶液，震荡混匀，室温放置5-10分钟。 2. 加入200 μL无水乙醇，涡旋震荡充分混匀。 注意：加入无水乙醇后可能会产生白色沉淀，不会影响后续实验。 3. 将步骤2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（Spin Columns DM）中，若一次不能加完溶液，可分多次转入。 12,000 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。 4. 向吸附柱中加入500 μL Buffer GW1 （使用前检查是否已加入无水乙醇）， 12,000 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 GW2（使用前检查是否已加入无水乙醇）， 12,000 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。 注意：如需进一步提高DNA纯度，可重复步骤5。 6. 12,000 rpm离心2分钟，倒掉收集管中废液。将吸附柱置于室温数分钟，以彻底晾干。 注意：这一步的目的是将吸附柱中残余的乙醇去除，乙醇的残留会影响后续的酶促反应（酶切、PCR等）。 7. 将吸附柱置于一个新的离心管（自备）中，向吸附柱的中间部位悬空加入50-200μL Buffer GE或灭菌水，室温放置2-5分钟， 12,000 rpm离心1分钟，收集DNA溶液， -20℃保存DNA。 注意： 1）如果下游实验对pH值或EDTA敏感，可以用灭菌水洗脱。洗脱液的pH值对洗脱效率有很 大影响，若用水做洗脱液应保证其pH值在7.0-8.5（可以用NaOH将水的pH值调到此范围），pH值 低于7.0时洗脱效率不高。 2） Buffer GE在65-70℃水浴预热，离心之前室温孵育5分钟可以增加产量；用另外的50-200μL Buffer GE或灭菌水再次洗脱可以增加产量。 3）如果要提高DNA的终浓度，可将得到的溶液重新加入到吸附柱中，室温放置2-5分钟，12,000 rpm离心1分钟；若洗脱体积小于200 μL，可以增加DNA的终浓度，但可能会减少总产量。如果DNA 的量小于1 μg，推荐用50 μL Buffer GE或灭菌水洗脱。 4）因为保存在水中的DNA会受到酸性水解作用的影响，如需长期保存，推荐用Buffer GE洗脱并于 -20℃保存。 Product content:
This reagent kit is suitable for extracting high-purity total DNA from various samples such as fresh or frozen animal tissues, cells, blood, bacteria, etc. This product can purify DNA fragments with a maximum molecular weight of 50 kb. The purification process does not require the use of toxic solvents such as phenol or chloroform, nor does it require ethanol precipitation. This reagent kit adopts an optimized buffer system to efficiently and specifically bind DNA from the lysis solution to the silica matrix centrifuge adsorption column. Inhibitors of PCR and other enzymatic reactions can be effectively removed through a two-step washing step. Finally, high-purity DNA can be obtained by washing with low salt buffer or water. The purified DNA can be directly used for downstream experiments such as enzyme digestion, PCR, Real Time PCR, library construction, Southern Blot, and molecular labeling. Self prepared reagent: anhydrous ethanol Enzymatic Lysis Buffer (preparation required for extracting genomic DNA from Gram positive bacteria). Self prepared reagent: Enzymatic Lysis Buffer Formula: 20 mM Tris, pH 8.0; 2 mM Na2 EDTA; 1.2% Triton self prepared reagent: X-100; Lysozyme with a final concentration of 20 mg/mL. Preparation and important precautions before the experiment: 1. Samples should avoid repeated freeze-thaw cycles, otherwise it may result in smaller extracted DNA fragments and a decrease in extraction volume. 2.If extracting the genome of bacterial cultures with a large accumulation of secondary metabolites or thick cell walls, it is recommended to collect samples early in the logarithmic growth phase. 3.Before the first use, anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the instructions on the reagent bottle label. 4. Before use, please check if there is any crystallization or precipitation in Buffer GTL and Buffer GL. If there is any crystallization or precipitation, please dissolve Buffer GL and Buffer GTL again in a 56 ℃ water bath. 5. If downstream experiments are sensitive to RNA contamination, 4 can be added before adding Buffer GL μ RNase A of L DNase Free (100 mg/mL) was not provided in this kit. Operation steps： Genome extraction from blood and cell samples 1. Material processing 1a If the extracted material is mammalian anticoagulant blood (non nucleated red blood cells), it can be directly directed to 50-200 μ Add Buffer GTL to fresh or frozen anticoagulant blood samples to supplement up to 200 μ L; 1b If the extracted material is anticoagulant blood from poultry, birds, amphibians, or lower level organisms, and their red blood cells are nucleated cells, take 5-10 μ L fresh or frozen anticoagulant blood samples, add Buffer GTL to supplement up to 200 μ L; 1c The cells cultured on the wall should be first processed into a cell suspension (with a maximum extraction amount of 5 × 10 cells), centrifuged at 2000 rpm (400 × g) for 5 minutes, discarded from the supernatant, and added with 200 μ L GTL, oscillate until the sample is completely suspended; Note: To remove RNA, add 4 after completing the above steps μ RNase A solution with a concentration of 100 mg/mL was vortexed for 15 seconds and left at room temperature for 2 minutes. 2. Add 20 μ L Protein K. 3. Add 200 μ L Buffer GL, vortex oscillation thoroughly mixed, 56 ℃ water bath for 10 minutes. 4. Temporarily centrifuge to remove water droplets from the inner wall of the tube cover. Join 200 μ L anhydrous ethanol, vortex and shake thoroughly to mix well. Short centrifugation. Attention: 1) After adding Bu ff er GL and anhydrous ethanol, immediately vortex shake and mix well. 2) The addition of Bu ff er GL and anhydrous ethanol may produce white precipitates, which will not affect subsequent experiments. Some organizations may form sol-gel products after adding Bu ff er GL and anhydrous ethanol, and it is recommended to perform severe shaking or vortex treatment at this time. 5. Add all the solutions obtained in the previous step to the spin columns DM that have been loaded into the collection tube. If the solution cannot be added at once, it can be transferred multiple times. Centrifuge at 12000 rpm (~13400 × g) for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. 6. Add 500 to the adsorption column μ L Buffer GW1 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. 7. Add 500 to the adsorption column μ L Buffer GW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. Note: To further improve DNA purity, repeat step 7. 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. Note: 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 in a new centrifuge tube (provided by oneself) and add 50-200 to the middle of the adsorption column in the air μ L Buffer GE or sterilized water, leave at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 1 minute, collect DNA solution, and store DNA at -20 ℃. Attention: 1) If downstream experiments are sensitive to pH or EDTA, they can be washed off with sterilized water. The pH value of the eluent has a significant impact on the elution efficiency. If water is used as the eluent, its pH value should be ensured to be between 7.0-8.5 (NaOH can be used to adjust the pH value of the water to this range). When the pH value is below 7.0, the elution efficiency is not high. 2) Preheating the GE in a water bath at 65-70 ℃ and incubating it at room temperature for 5 minutes before centrifugation can increase yield; Use an additional 50-200 μ Re washing with GE or sterilized water can increase yield. 3) If the final concentration of DNA needs to be increased, the obtained solution can be re added to the adsorption column, left at room temperature for 2-5 minutes, and centrifuged at 12000 rpm for 1 minute; If the elution volume is less than 200 μ L. It is possible to increase the final concentration of DNA, but it may reduce the total yield. If the amount of DNA is less than 1 μ g. Recommended 50 μ Wash with GE or sterilized water. 4) Because DNA stored in water is affected by acidic hydrolysis, if long-term preservation is required, it is recommended to elute with Bu ff er GE and store at -20 ℃. Genome extraction from animal tissues 1. Material processing If the extracted material is animal tissue, take 25 mg (the amount of spleen tissue should be less than 10 mg); If the material is mouse tail, take a section of rat tail with a length of 0.4-0.6 cm or two sections of mouse tail with a length of 0.4-0.6 cm. 1a. After liquid nitrogen grinding or cutting the sample into small pieces, place it in a 1.5 mL centrifuge tube and add 180 mL μ Label different samples with L Buffer GTL. 1b If using a homogenizer to process the sample, add no more than 80% of the homogenizer to the sample before homogenization μ L Buffer GTL, add 100 after homogenization μ L Buffer GTL. Attention: 1) Ensure that the quantity of each organization does not exceed the recommended range. 2) The tissue samples can be ground with liquid nitrogen or homogenized with a homogenizer before adding Bu ff er GTL, which can increase the cracking efficiency. 2. Add 20 μ L Protein K, vortex oscillation thoroughly mixes the sample. Take a 56 ℃ water bath until the tissue is completely lysed. During the incubation process, the centrifuge tube can be inverted or shaken periodically to disperse the sample. Attention: 1) The digestion time varies for different tissues, usually taking 1-3 hours to complete. The tail of the mouse needs to be digested for 6-8 hours, and if necessary, overnight digestion will not affect subsequent operations. 2) If there is still gel like substance after incubation and vortex oscillation, extend the incubation time at 56 ℃ or add another 20 μ L Protein K digestion. 3) To remove RNA, add 4 after completing the above steps μ RNase A solution with a concentration of 100 mg/mL, vortex for 15 seconds, and leave at room temperature for 5-10 minutes. 3. Add 200 μ L Buffer GL, vortex shake thoroughly and mix well, take a water bath at 70 ℃ for 10 minutes. Add 200 after brief centrifugation μ L anhydrous ethanol, vortex and shake thoroughly to mix well. Attention: 1) After adding Bu ff er GL and anhydrous ethanol, immediately vortex and shake to mix well. 2) The addition of Bu ff er GL and anhydrous ethanol may produce white precipitates, which will not affect subsequent experiments. Some tissues (such as the spleen and lungs) may form sol-gel products after adding Bu ff er GL and anhydrous ethanol. In this case, it is recommended to perform vigorous shaking or vortex treatment. 4. Centrifuge briefly and add all the solution obtained in step 3 to the spin columns DM that have been loaded into the collection tube. If the solution cannot be added at once, it can be transferred multiple times. Centrifuge at 12000 rpm (~13400 × g) for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. 5. Add 500 to the adsorption column μ L Buffer GW1 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. 6. Add 500 to the adsorption column μ L Buffer GW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. Note: To further improve DNA purity, repeat step 6. 7.12000 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. Note: 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.). 8. Place the adsorption column in a new centrifuge tube (provided by oneself) and add 50-200 to the middle of the adsorption column in the air μ L Buffer GE or sterilized water, leave at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 1 minute, collect DNA solution, and store DNA at -20 ℃. Attention: 1) If downstream experiments are sensitive to pH or EDTA, they can be washed off with sterilized water. The pH value of the eluent has a significant impact on the elution efficiency. If water is used as the eluent, its pH value should be ensured to be between 7.0-8.5 (NaOH can be used to adjust the pH value of the water to this range). When the pH value is below 7.0, the elution efficiency is not high. 2) Preheating the GE in a water bath at 65-70 ℃ and incubating it at room temperature for 5 minutes before centrifugation can increase yield; Use an additional 50-200 μ Re washing with GE or sterilized water can increase yield. 3) If the final concentration of DNA needs to be increased, the obtained solution can be re added to the adsorption column, left at room temperature for 2-5 minutes, and centrifuged at 12000 rpm for 1 minute; If the elution volume is less than 200 μ L. It is possible to increase the final concentration of DNA, but it may reduce the total yield. If the amount of DNA is less than 1 μ g. Recommended 50 μ Wash with GE or sterilized water. 4) Because DNA stored in water is affected by acidic hydrolysis, if long-term preservation is required, it is recommended to elute with Bu ff er GE and store at -20 ℃. i ii Genomic extraction of blood and cell samples 1. Bacterial sample pretreatment 1a Gram negative bacteria (1) Take 1-5mL of bacterial culture (10 ^ -10 ^ cells, up to a maximum of 2 × 10 ^ cells) and place it in a centrifuge tube (self prepared). Centrifuge at 12000 rpm (~13400 × g) for 1 minute and try to aspirate the supernatant as much as possible. (2) Add 180 to the precipitate μ L Buffer GTL, shake to suspend bacterial weight. (3) Join 20 μ L Protein K, vortex mix well, incubate at 56 ° C until the bacterial cell is completely lysed, and during the incubation process, invert or shake the centrifuge tube periodically to disperse the sample. Note: To remove RNA, add 4 after completing the above steps μ L RNase A solution with a concentration of 100 mg/mL, shake well and let stand at room temperature for 5-10 minutes. (4) Join 200 μ L Buffer GL, vortex oscillation mixing. 1b Gram positive bacteria (1) Take 1-5 mL of bacterial culture (10 ^ -10 ^ cells, maximum not exceeding 2 x 10 ^ cells) and place it in a centrifuge tube (self prepared). Centrifuge at 12000 rpm for 1 minute and try to aspirate the supernatant as much as possible. (2) Join 180 μ L Enzymatic Lysis Buffer (self provided) suspends the bacterial weight. (3) Incubate at 37 ℃ for 30 minutes. (4) Join 20 μ L Protein K vortex oscillation, thoroughly mixed. Join 200 μ L Buffer GL, vortex oscillation mixing. Incubate at 56 ℃ for 30 minutes. Attention: 1) If necessary, incubation at 95 ° C for 15 minutes can inactivate the pathogen, but incubation at 95 ° C can cause some DNA degradation. 2) To remove RNA, add 4 after completing the above steps μ L RNase A solution with a concentration of 100 mg/mL, shake well and let stand at room temperature for 5-10 minutes. 2. Add 200 μ L anhydrous ethanol, vortex and shake thoroughly to mix well. Attention: Adding anhydrous ethanol may produce white precipitates, which will not affect subsequent experiments. 3. Add all the solution obtained from step 2 (including the formed precipitate) to the adsorption column (Spin Columns DM) that has been loaded into the collection tube. If the solution cannot be added at once, it can be transferred multiple times. Centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. 4. Add 500 to the adsorption column μ L Buffer GW1 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. 5. Add 500 to the adsorption column μ L Buffer GW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. Note: To further improve DNA purity, repeat step 5. 6.12000 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. Note: 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.). 7. Place the adsorption column in a new centrifuge tube (provided by oneself) and add 50-200 to the middle of the adsorption column in the air μ L Buffer GE or sterilized water, leave at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 1 minute, collect DNA solution, and store DNA at -20 ℃. Attention: 1) If downstream experiments are sensitive to pH or EDTA, they can be washed off with sterilized water. The pH value of the eluent has a significant impact on the elution efficiency. If water is used as the eluent, its pH value should be ensured to be between 7.0-8.5 (NaOH can be used to adjust the pH value of the water to this range). When the pH value is below 7.0, the elution efficiency is not high. 2) Preheating the GE in a water bath at 65-70 ℃ and incubating it at room temperature for 5 minutes before centrifugation can increase yield; Use an additional 50-200 μ Re washing with GE or sterilized water can increase yield. 3) If the final concentration of DNA needs to be increased, the obtained solution can be re added to the adsorption column, left at room temperature for 2-5 minutes, and centrifuged at 12000 rpm for 1 minute; If the elution volume is less than 200 μ L. It is possible to increase the final concentration of DNA, but it may reduce the total yield. If the amount of DNA is less than 1 μ g. Recommended 50 μ Wash with GE or sterilized water. 4) Because DNA stored in water is affected by acidic hydrolysis, if long-term preservation is required, it is recommended to elute with Bu ff er GE and store at -20 ℃. |
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