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| 英文名称 | Bacteria 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 |
产品简介 本试剂盒适用于从革兰氏阴性菌、革兰氏阳性菌中提取高纯度总DNA，每次可处理106-108个细胞，获得多至20 μg的总DNA。纯化过程中不需苯酚或氯仿等有毒溶剂， 无需乙醇沉淀，一小时内即可获得高纯度DNA。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，而其他污染物可流过膜，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，即可获得高纯度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；提取革兰氏阳性菌需自备Enzymatic Lysis Buffer。 Enzymatic Lysis Buffer配制方法：20 mM Tris，pH8.0；2 mM Na2-EDTA，pH8.0；1.2% Triton X-100。121℃灭菌处理20分钟，加入适量的Lysozyme（溶菌酶）其终浓度为20 mg/ml。 实验前准备及重要注意事项 1. 向Proteinase K中加入1.25 ml Proteinase K Storage Buffer使其溶解，-20℃保存。配制好的Proteinase K勿长时间室温放置，避免反复冻融，以免影响其活性。 2. 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。 3. 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。 4. 第一次使用前应按照试剂瓶标签的说明在Buffer GW1和Buffer GW2中加入无水乙醇。 5. 使用前请检查Buffer GTL和Buffer GL是否出现结晶或者沉淀，如有结晶或者沉淀出现，请将Buffer GL和Buffer GTL于56℃水浴重新溶解。 6. 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。 如果提取样品为革兰氏阳性菌，需客户自行配制Enzymatic Lysis Buffer处理菌体，其中需使用浓度为20 mg/ml的Lysozyme（溶菌酶），Lysozyme本试剂盒并未提供。 操作步骤 ⅰ革兰氏阴性菌基因组DNA的提取 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（~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，直至溶液变清亮，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，涡旋震荡充分混匀。加200 μl无水乙醇，涡旋震荡充分混匀。 短暂离心，使管壁上的溶液收集到管底。 注意：1）如果多个样品一起操作，Buffer GL和无水乙醇可以等比例混匀后一起加入，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。 5. 将步骤4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，弃废液，将吸附柱放回收集管中。 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，倒掉收集管中的废液。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，乙醇残留会影响后续酶促反应（酶切、PCR等）。 9．将吸附柱置于一个新的离心管中，向吸附柱的中间部位悬空加入50-200 μl Buffer GE，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） 离心之前室温孵育5分钟可以增加产量。 3） 用另外的50-200 μl Buffer GE或灭菌水再次洗脱可以增加产量。 4） 如果要提高DNA的终浓度，可以将步骤9所得的DNA洗脱液重新加至吸附膜上，重复步骤9；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。 5） 保存在水中的DNA会受到酸性水解作用影响，如需长期保存，推荐用Buffer GE洗脱并于-20℃保存。 ⅰⅰ革兰氏阳性菌基因组DNA的提取 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（~13,400×g）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。 Enzymatic Lysis Buffer配制方法见说明书前部分自备试剂。 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。 注意：不要把Proteinase K直接加到Buffer GL中。 5．56℃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。 6. 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。 7. 将步骤6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），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。 8. 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。 9. 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9。 10.12,000 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。 注意：这一步目的是将吸附柱中残余的乙醇去除，乙醇残留会影响后续酶促反应（酶切、PCR等）。 11.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50-200 μl Buffer GE，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） 离心之前室温孵育5分钟可以增加产量。 3） 用另外的50-200 μl Buffer GE或灭菌水再次洗脱可以增加产量。 4） 如果要提高DNA的终浓度，可以将步骤11所得的DNA洗脱液重新加至吸附膜上，重复步骤11； 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。 5） 保存在水中的DNA会受到酸性水解作用影响，如需长期保存，推荐用Buffer GE洗脱并于-20℃保存。
Products This kit is suitable for extracting high purity total DNA from Gram-negative and Gram-positive bacteria. 106-108 cells can be processed at a time, and up to 20 μg of total DNA can be obtained within one hour without the need for toxic solvents such as phenol or chloroform, and without the need for ethanol precipitation. The optimized buffer system enables the DNA in the lysate to be efficiently and specifically bound to the silica matrix centrifugal adsorption column, while other contaminants can flow through the membrane, and the inhibitors of PCR and other enzymatic reactions can be effectively removed through a two-step washing step, and finally washed off with low-salt buffer or water, so that high-purity DNA can be obtained.The purified DNA can be used for downstream experiments such as digestion, PCR, Real-Time PCR, library construction, Southern Blot and molecular labeling, molecular labeling and other downstream experiments. Self-contained reagents: anhydrous ethanol; Enzymatic Lysis Buffer is required for extraction of Gram-positive bacteria. Enzymatic Lysis Buffer was prepared by 20 mM Tris, pH 8.0; 2 mM Na2-EDTA, pH 8.0; and 1.2% Triton X-100. 121°C sterilization for 20 minutes, and the appropriate amount of Lysozyme was added at a final concentration of 20 mg/ml. Pre-experiment Preparation and Important Notes 1. Add 1.25ml Proteinase K Storage Buffer to Proteinase K to dissolve it and store it at -20℃. Do not leave the prepared Proteinase K at room temperature for a long time, and avoid repeated freezing and thawing to avoid affecting its activity. 2. Repeated freezing and thawing of the sample should be avoided, as this may result in smaller DNA fragments and a decrease in the amount of extracted DNA. 3. If extracting genomes from bacterial cultures with high accumulation of secondary metabolites or thick cell walls, it is recommended that samples be collected early in the logarithmic phase. 4. Anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the instructions on the label of the reagent bottle before first use. 5. Before use, please check Buffer GTL and Buffer GL for crystallization or precipitation. If crystallization or precipitation occurs, please re-dissolve Buffer GL and Buffer GTL in a 56℃ water bath. 6. If the downstream experiments are sensitive to RNA contamination, 4μl of DNase-Free RNase A (100mg/ml) can be added before adding Buffer GL. RNase A is not provided in this kit. If the extracted samples are Gram-positive bacteria, customers need to prepare their own Enzymatic Lysis Buffer to treat the bacteria, which requires the use of Lysozyme (lysozyme) at a concentration of 20 mg/ml, which is not provided in this kit. Procedure i Extraction of genomic DNA from Gram-negative bacteria 1. Take 1-5 ml of bacterial culture (106-108 cells, maximum 2×109 cells) and put it into a centrifuge tube (provided), centrifuge it at 12,000 rpm (~13,400×g) for 1 minute, and aspirate the supernatant as much as possible. 2. Add 180 μl Buffer GTL to the precipitate and shake to resuspend the bacteria. 3. Add 20 μl of Proteinase K, vortex and mix well, incubate at 56°C until the solution becomes clear, and invert or shake the centrifuge tube at intervals during the incubation to disperse the sample. Note: If RNA removal is required, add 4 μl of RNase A solution at a concentration of 100 mg/ml after the above steps are completed, shake to mix, and leave for 5-10 minutes at room temperature. 4. Add 200μl Buffer GL and mix well with vortexing and shaking. Add 200μl of anhydrous ethanol and mix well with vortexing and shaking. Centrifuge briefly so that the solution on the walls of the tube collects at the bottom. Note: 1) If multiple samples are manipulated together, Buffer GL and anhydrous ethanol can be mixed in equal proportions and then added together, shaking to mix. 2) The addition of Buffer GL and anhydrous ethanol may produce a white precipitate that will not affect subsequent experiments. 5. Add all of the solution obtained in step 4 (including the precipitate formed) to the Spin Columns DM in the collection tube, or if the solution cannot be added all at once, transfer it several times. centrifuge at 12,000 rpm for 1 minute, discard the waste solution, and return the column to the collection tube. 6. Add 500 μl of Buffer GW1 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and return the adsorption column to the collection tube. 7. Add 500 μl of Buffer GW2 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube. Note: Step 7 can be repeated if further DNA purity is required. 8. Centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in the collection tube. Leave the adsorbent column at room temperature for several minutes to 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.). 9. Place the adsorption column in a new centrifuge tube, add 50-200 μl Buffer GE to the middle part of the adsorption column overhanging the center of the adsorption column, leave it at room temperature for 2-5 minutes, centrifuge it at 12,000 rpm for 1 minute, collect the DNA solution, and store the DNA at -20 ℃. note: 1) If the downstream experiments are sensitive to the pH or EDTA, the elution can be done with sterilized water. The pH of the elution solution has a great influence on the elution efficiency. If water is used as the elution solution it should be ensured that its pH is 7.0-8.5 (the pH of water can be adjusted to this range with NaOH), and the elution efficiency is not high when the pH is lower than 7.0. 2) Incubation at room temperature for 5 minutes prior to centrifugation increases yield. 3) Re-elution with an additional 50-200 μl Buffer GE or sterilized water can increase the yield. 4) If the final concentration of DNA is to be increased, the DNA eluate obtained in step 9 can be re-spiked onto the adsorbent membrane and step 9 repeated; if the elution volume is less than 200 μl, the final concentration of DNA can be increased, but the total yield may be reduced. If the amount of DNA is less than 1 μg, elution with 50 μl Buffer GE or sterilized water is recommended. (5) 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℃. i. Extraction of genomic DNA from Gram-positive bacteria 1. Take 1-5 ml of bacterial culture (106-108 cells, maximum 2×109 cells) and put it into a centrifuge tube (provided), centrifuge it at 12,000 rpm (~13,400×g) for 1 minute, and aspirate the supernatant as much as possible. 2. Add 180μl Enzymatic Lysis Buffer (self-provided) to resuspend the bacteria. Enzymatic Lysis Buffer is prepared as described in the Self-Prepared Reagents section in the front of the manual. 3. Incubate at 37°C for 30 minutes. 4. Add 20μl Proteinase K and mix well. Add 200μl of Buffer GL and mix well with vortexing and shaking. Note: Do not add Proteinase K directly to Buffer GL. Incubate at 5.56°C for 30 minutes. Note: 1) If desired, incubation at 95°C for 15 minutes will inactivate the pathogen, but 95°C incubation will cause some DNA degradation. (2) If RNA removal is required, add 4μl of RNase A solution at a concentration of 100mg/ml after the above steps are completed, shake and mix well, and leave for 5-10 minutes at room temperature. 6. Add 200μl of anhydrous ethanol and mix well with vortex shaking. Note: The addition of anhydrous ethanol may produce a white precipitate that will not affect subsequent experiments. 7. Add all of the solution obtained in step 6 (including the precipitate formed) to the Spin Columns DM that have been loaded into the collection tube, and if the solution cannot be added all at once, it can be transferred in several times. centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid from the collection tube, and put the column back into the collection tube. 8. Add 500 μl of Buffer GW1 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube. 9. Add 500 μl Buffer GW2 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge the column at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the column back into the collection tube. Note: Step 9 can be repeated if further DNA purity is required. 10. Centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in the collection tube. Leave the adsorption column at room temperature for several minutes to dry thoroughly. Note: The purpose of this step is to remove residual ethanol from the adsorption column; ethanol residue can interfere with subsequent enzymatic reactions (digestion, PCR, etc.). 11. Place the adsorption column in a new centrifuge tube (self-provided), add 50-200 μl of Buffer GE to the center of the adsorption column overhanging the center of the adsorption column, let it stand at room temperature for 2-5 minutes, centrifuge at 12,000 rpm for 1 minute, collect the DNA solution, and store the DNA at -20℃. Note: 1) If the downstream experiment is sensitive to pH or EDTA, you can use sterilized water for elution. The pH of the eluent has a great influence on the elution efficiency, if water is used as the eluent should ensure that its pH is 7.0-8.5 (you can use NaOH to adjust the pH of the water to this range), and the elution efficiency is not high when the pH is lower than 7.0. 2) Incubation at room temperature for 5 minutes prior to centrifugation increases yield. 3) Re-elution with an additional 50-200 μl Buffer GE or sterilized water can increase the yield. 4) If the final concentration of DNA is to be increased, the DNA eluate obtained in step 11 can be re-spiked onto the adsorbent membrane and step 11 repeated; if the elution volume is less than 200 μl, the final concentration of DNA can be increased, but the total yield may be reduced. If the amount of DNA is less than 1 μg, elution with 50 μl Buffer GE or sterilized water is recommended. (5) 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℃. |
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| 1. Dangfeng Wang, Xin Wang, Shanshan Zhou, Likun Ren, Yuqiong Meng, Rui Ma, Shulin Wang, Zhiteng Liu, Abdulhakeem S. Alamri, Majid Alhomrani, Zihui Zhang, Fangchao Cui, Tingting Li, Jianrong Li. (2024) Radish residue carbon dots-based novel starch/chitosan film with high antioxidant, biocompatibility, and antibacterial activities for salmon fillets' active packaging. INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, 273 (133107). [PMID:38897524] [10.1016/j.ijbiomac.2024.133107] |