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| 英文名称 | Viral DNA/RNA 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 本试剂盒适用于从新鲜或冷冻的血浆、血清和无细胞体液中提取病毒RNA和DNA。 本品无需使用苯酚、氯仿等有机溶剂抽提，操作简单。本试剂盒采用独特的缓冲体系使裂解液中的病毒核酸高效特异地结合在硅胶质离心吸附柱上，PCR和酶反应的抑制剂以及残留的杂质可通过两步有效的漂洗步骤有效去除，最后使用低盐缓冲液或水洗脱，即可获得高纯度的病毒核酸。纯化的病毒核酸不含蛋白、核酸酶和其他杂质，可直接用于PCR、RT-PCR、Real-Time PCR、印迹实验等。 自备试剂：无水乙醇。 实验前及重要注意事项 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。勿将Proteinase K直接加入到Buffer GL中。 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. 血清或血浆避免反复冻融，反复冻融会导致蛋白变性或产生沉淀，减少病毒滴度进 而影响提取病毒核酸的产量。 4. 第一次使用前应按照试剂瓶标签说明在Buffer GW1和Buffer GW2中加入无水乙醇。 5. 使用前请检查Buffer GL是否出现结晶或者沉淀，如有结晶或者沉淀，请将Buffer GL于56℃水浴重新溶解。 操作步骤 1. 取1.5 ml离心管（自备），加入20 μl Proteinase K。 2. 向离心管中加入200 μl血清或血浆。加入200 μl Buffer GL，涡旋震荡15秒。注意：1）样本体积不足200μl可以加入0.9% NaCl（自备）补足。2）为确保样本有效裂解，加入Buffer GL后，需将样本与Buffer GL充分混匀。 3．56℃孵育15分钟，短暂离心，将管壁上的溶液收集到管底。 4. 加入250μl无水乙醇，涡旋震荡15秒，室温放置5分钟，短暂离心，将管壁上的溶液收集到管底。 注意：如果环境温度超过25℃，无水乙醇应在冰上预冷后使用。 5. 将步骤4中所得溶液加入到已装入收集管的吸附柱（RNase-Free Columns RS）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. 向吸附柱中加入500 μl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。 9.12 ,000 rpm离心3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：这一步的目的是吸附柱中残余的乙醇去除，乙醇残留会影响后续的酶促反应（酶切、PCR等）。 10.将吸附柱置于一个新的收集管（RNase-Free Centrifuge Tube ）中，向吸附柱膜的中间部位悬空加入20-150 μl Buffer RE或灭菌水，室温放置2-5分钟，12,000 rpm离心1分钟，收集核酸溶液。 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） 如需长期保存，请将DNA溶液置于-20℃保存，RNA溶液置于-70℃保存。 3） 如果要提高DNA/RNA的终浓度，可以将步骤10所得的DNA/RNA洗脱液重新加至吸附膜上，重复步骤10。
Products This kit is suitable for the extraction of viral RNA and DNA from fresh or frozen plasma, serum and cell-free body fluids. It is easy to operate as it does not require the use of organic solvents such as phenol and chloroform for extraction. The kit uses a unique buffer system to enable efficient and specific binding of viral nucleic acids in lysate to silica gel centrifugal adsorption columns. Inhibitors of PCR and enzyme reactions as well as residual impurities can be efficiently removed in a two-step effective rinsing step, and finally high purity viral nucleic acids can be obtained by using a low-salt buffer or water for elution. The purified viral nucleic acid is free of protein, nuclease and other impurities, and can be used directly in PCR, RT-PCR, Real-Time PCR, blotting experiments and so on. Self-contained reagent: anhydrous ethanol. Pre-experiment 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. Do not add Proteinase K directly into Buffer GL. 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. Avoid repeated freezing and thawing of serum or plasma, which can lead to protein denaturation or precipitation, reducing the viral titer and thus affecting the yield of extracted viral nucleic acids. 4. Anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the label instructions of the reagent bottle before first use. 5. Check Buffer GL for crystallization or precipitation before use. If crystallization or precipitation occurs, redissolve Buffer GL in a water bath at 56℃. Procedure 1. Take a 1.5 ml centrifuge tube (self-provided) and add 20 μl Proteinase K. 2. Add 200 μl serum or plasma to the centrifuge tube. Add 200μl Buffer GL and vortex and shake for 15 seconds. Note: 1) Sample volume less than 200 μl can be made up by adding 0.9% NaCl (self-provided). 2) In order to ensure effective lysis of the sample, the sample needs to be mixed well with Buffer GL after adding Buffer GL. 3. Incubate at 56°C for 15 minutes, centrifuge briefly, and collect the solution from the wall of the tube to the bottom of the tube. 4. 250 μl of anhydrous ethanol was added, vortexed and shaken for 15 seconds, left at room temperature for 5 minutes, centrifuged briefly, and the solution on the wall of the tube was collected at the bottom of the tube. Note: If the ambient temperature exceeds 25°C, anhydrous ethanol should be used after pre-cooling on ice. 5. Add the solution obtained in step 4 to the adsorbent column (RNase-Free Columns RS) that has been loaded into the collection tube, and if the solution cannot be added at one time, it can be transferred in several times. centrifuge the column at 12,000 rpm (~13,400 × g) for 1 min, pour off the waste liquid in the collection tube, and put the column back into 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 put the adsorption column back into 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. Add 500 μl of anhydrous ethanol to the adsorbent column and 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. 9. Centrifuge at 12,000 rpm for 3 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 the removal of residual ethanol from the adsorbent column; ethanol residue can interfere with subsequent enzymatic reactions (digestion, PCR, etc.). 10. Place the adsorption column in a new collection tube (RNase-Free Centrifuge Tube), add 20-150 μl of Buffer RE or sterilized water overhanging the middle of the adsorption column membrane, leave it at room temperature for 2-5 minutes, and then centrifuge it at 12,000 rpm for 1 minute to collect the nucleic acid solution. 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 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) For long-term storage, please store the DNA solution at -20℃ and the RNA solution at -70℃. 3) If the final concentration of DNA/RNA is to be increased, the DNA/RNA eluate obtained in step 10 can be re-spiked onto the adsorbent membrane and step 10 repeated. |
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