首页
400-620-6333
计算溶液所需的质量、体积或浓度。
动物组织/细胞RNA提取试剂盒（DNase I）
有货
库存信息
| 货号 (SKU) | 包装规格 | 是否现货 | 价格 | 数量 |
|---|---|---|---|---|
| R669990-50T |
50T |
期货  |
|
切换导航
关闭
搜索
基本描述
| 英文名称 | RNApure Tissue&Cell Kit (DNase I) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 储存温度 | 室温,-20°C储存 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 运输条件 | 超低温冰袋运输 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 产品介绍 |
产品内容
产品简介 本试剂盒将高效的异硫氰酸胍裂解技术与硅基质膜纯化技术相结合，可从动物细胞及组织中高效提取总RNA。起始样本一般最多30 mg组织或1x107细胞。本试剂盒还可回收未完全纯化的RNA、体外转录和酶促反应后得到的RNA。用本试剂盒可提取纯化分子量大于200碱基的高品质RNA，几乎无DNA残留。如果要进行对微量DNA非常敏感的RNA实验，残留的DNA可利用无RNase的DNase在柱上进行消化去除。提取的RNA可用于RT-PCR、Nothern Blot、Dot Blot等下游实验。 自备试剂：β-巯基乙醇、无水乙醇（新开封或提取RNA专用）。 实验前准备及重要注意事项 1. 预防RNase污染，应注意以下几方面： 1） 使用无RNase的塑料制品和枪头，避免交叉污染。 2） 配制溶液应使用无RNase的水。 3） 操作人员戴一次性口罩和手套，实验过程中要勤换手套。 2. 提取的样品避免反复冻融，否则影响RNA提取的量和质量。 3. Buffer RL在使用前请加入β-巯基乙醇，1ml Buffer RL加10μl β-巯基乙醇。加入β-巯基乙醇的Buffer RL室温可保存1个月。 4. 第一次使用前应按照试剂瓶标签的说明先在Buffer RW2中加入无水乙醇。 5. Buffer RL如果产生沉淀，可于56℃加热使其溶解后室温放置。 所有离心步骤均在室温下进行，且所有操作步骤动作要迅速。 操作步骤 1. 样品处理 1a 组织：将组织在液氮中磨碎。每20-30 mg组织加600 μl Buffer RL（使用前检查是否加入β-巯基乙醇），组织样本少于20 mg加350 μl Buffer RL。样品体积不超过Buffer RL体积的十分之一。 1b 单层培养细胞：将细胞在培养瓶中直接裂解或处理成细胞悬液，离心得到细胞沉淀， 弃上清，每6-10 cm2培养面积加入600 μl Buffer RL，小于6 cm2加入350 μl Buffer RL， 反复吹打几次，使其充分裂解。 1c 细胞悬液：12,000 rpm（～13,400×g）离心1分钟弃上清，得到细胞沉淀。每5×106-1×107细胞加入600 μl Buffer RL，少于5×106细胞加入350 μl Buffer RL，反复吹打几次，使其充分裂解。 注意：1）尽量除尽细胞培养基，细胞培养基可能抑制细胞的裂解影响RNA产量。 2）尽量使细胞充分悬浮并充分裂解，否则影响RNA产量。 2. 样品充分裂解后，室温放置5分钟，使蛋白核酸复合物完全分离。 3．12,000 rpm离心2-5min，取上清进行以下操作。 4. 向步骤3中得到的溶液中加入1倍体积（600 μl 或350 μl）的70%乙醇（无RNase水配制），混匀。 注意：加入乙醇后可能会产生沉淀，不会影响后续实验。 5. 将上步所得溶液全部加入到已装入收集管的吸附柱（Spin Columns RM）中，若一次不能将全部溶液加入吸附柱中，请分两次转入，12,000 rpm离心1分钟，弃废液。将吸附柱放回收集管中。 注意：吸附柱的最大载量为100 µg，不要超载，否则会影响RNA的产量和纯度。 6. 向吸附柱中加入350 μl Buffer RW1，12,000 rpm离心1分钟，弃废液，将吸附柱重新放回收集管中。 7. 配制DNase I 混合液：取52 μl RNase-Free Water，向其中加入8 μl 10×Reaction Buffer和20 μl DNase I（1 U/μl），混匀， 配制成终体积为80 μl的反应液。 8. 向吸附柱中直接加入80 µl DNase I 混合液，20-30℃孵育15分钟。 9. 向吸附柱中加入200 μl Buffer RW1，12,000 rpm离心1分钟，弃废液，将吸附柱重新放回收集管中。 10. 向吸附柱中加入500 μl Buffer RW2（使用前检查是否加入无水乙醇）, 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。 11.重复步骤10。 12.12,000 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，以彻底晾干吸附柱中的无水乙醇。 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）。 13. 将吸附柱转入新的离心管中，向吸附膜中间位置加入30-50 μl RNase-Free Water，室温放置1分钟，12,000 rpm离心1分钟，收集RNA溶液，-70℃保存RNA，防止降解。注意：1）RNase-Free Wate体积不应小于30 μl，体积过小影响回收率。 2） 如果要提高RNA的产量，可用30-50 μl新的RNase-Free Water重复步骤13。 3） 如果要提高RNA浓度，可将得到的溶液重新加入到吸附柱中，重复步骤13。 Product Content
Products This kit combines highly efficient guanidine isothiocyanate cleavage technology with silica matrix membrane purification for the efficient extraction of total RNA from animal cells and tissues, typically up to 30 mg of tissue or 1x107 cells as a starting sample. The kit also allows recovery of incompletely purified RNA, in vitro transcription and RNA from enzymatic reactions. high quality RNA with molecular weights greater than 200 bases can be extracted and purified using the kit with virtually no DNA residue. If RNA experiments that are very sensitive to trace DNA are to be performed, residual DNA can be removed by on-column digestion using RNase-free DNase. The extracted RNA can be used in downstream experiments such as RT-PCR, Nothern Blot and Dot Blot. Self-contained reagents: β-mercaptoethanol, anhydrous ethanol (freshly opened or for RNA extraction). Pre-experiment Preparation and Important Notes 1. To prevent RNase contamination, attention should be paid to the following aspects: 1) Use RNase-free plastics and tips to avoid cross-contamination. 2) RNase-free water should be used to prepare the solution. (3) 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 amount and quality of RNA extraction. 3. Please add β-mercaptoethanol to Buffer RL before use, add 10μl of β-mercaptoethanol to 1ml of Buffer RL. Buffer RL with β-mercaptoethanol can be stored for 1 month at room temperature. 4. Anhydrous ethanol should be added to Buffer RW2 before first use according to the instructions on the reagent bottle label. 5. Buffer RL may be heated at 56°C to dissolve if precipitation occurs and then left at room temperature. All centrifugation steps are performed at room temperature and all maneuvers are performed quickly. Procedure 1. Sample handling 1a Tissue: Grind tissue in liquid nitrogen. Add 600 μl Buffer RL for every 20-30 mg of tissue (check for addition of β-mercaptoethanol before use), and 350 μl Buffer RL for tissue samples of less than 20 mg. Sample volume is not to exceed one-tenth of the Buffer RL volume. 1b Cells in monolayer culture: Lysed or processed into cell suspension directly in culture flask, centrifuged to obtain cell precipitate, discarded the supernatant, added 600μl Buffer RL for every 6-10 cm2 of culture area, 350μl Buffer RL for less than 6cm2, and blown several times repeatedly to make the cells lysed sufficiently. 1c Cell suspension: centrifuge at 12,000 rpm (~13,400 × g) for 1 min and discard the supernatant to obtain the cell precipitate. Add 600 μl Buffer RL for every 5×106-1×107 cells, and 350 μl Buffer RL for less than 5×106 cells, and blow several times repeatedly to fully lysate. Note: 1) Try to get rid of the cell culture medium, which may inhibit cell lysis affecting RNA yield. 2) Try to keep the cells well suspended and well lysed, otherwise RNA yield is affected. 2. After the sample is fully lysed, leave it at room temperature for 5 minutes to allow complete separation of the protein-nucleic acid complex. 3. Centrifuge at 12,000 rpm for 2-5 min and remove the supernatant for the following operations. 4. Add 1x volume (600μl or 350μl) of 70% ethanol (prepared without RNase water) to the solution obtained in step 3 and mix well. Note: The addition of ethanol may produce a precipitate that will not affect subsequent experiments. 5. Add all of the solution obtained in the previous step to the Spin Columns RM in the collection tube. If you cannot add all of the solution to the column at once, transfer it in two passes, centrifuge at 12,000 rpm for 1 minute, and discard the waste solution. Place the column back into the collection tube. Note: The maximum loading capacity of the adsorption column is 100µg, do not overload as this will affect the yield and purity of the RNA. 6. Add 350 μl Buffer RW1 to the adsorbent column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid and put the adsorbent column back into the collection tube. 7. Preparation of DNase I mixture: Take 52 μl of RNase-Free Water, add 8 μl of 10×Reaction Buffer and 20 μl of DNase I (1 U/μl) to it, mix well, and prepare a final volume of 80 μl of reaction solution. 8. Add 80µl of DNase I mixture directly to the adsorption column and incubate at 20-30°C for 15 minutes. 9. Add 200 μl Buffer RW1 to the adsorbent column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid and put the adsorbent column back into the collection tube. 10. Add 500μl Buffer RW2 to the column (check that anhydrous ethanol is added before use), centrifuge 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. 11. Repeat step 10. 12. 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 a few minutes to thoroughly dry the anhydrous ethanol in the adsorption column. Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can interfere with subsequent enzymatic reactions (digestion, PCR, etc.). 13. Transfer the adsorbent column into a new centrifuge tube, add 30-50 μl of RNase-Free Water to the middle of the adsorbent membrane, leave it at room temperature for 1 min, centrifuge at 12,000 rpm for 1 min, collect the RNA solution, and store the RNA at -70°C to prevent degradation. Note: 1) The volume of RNase-Free Wate 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 13 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 13 repeated. |
AI解读
安全和危险性（GHS）
技术规格说明书
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: