首页
400-620-6333
计算溶液所需的质量、体积或浓度。
金牌超量无内毒素质粒中提试剂盒
有货
库存信息
| 货号 (SKU) | 包装规格 | 是否现货 | 价格 | 数量 |
|---|---|---|---|---|
| G665573-10T |
10T |
期货  |
|
切换导航
关闭
搜索
基本描述
| 英文名称 | GoldHi EndoFree Plasmid Midi Kit | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 储存温度 | 室温 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 运输条件 | 常规运输 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 产品介绍 |
产品简介 本试剂盒专门用于从15-50 ml菌液中高效、快速提取质粒。在碱裂解法裂解细胞的 基础上，采用独特的硅基质膜吸附技术，高效专一的结合质粒DNA，每个吸附柱最高 可吸附250 μg的质粒DNA；同时采用特殊的缓冲液系统和除内毒素过滤器，有效去除 内毒素、基因组DNA、RNA、蛋白等杂质。由本试剂盒所得质粒纯度高、质量稳定， 可用于细胞转染，同时也可用于DNA测序，PCR，体外转录，内切酶消化等实验。 自备试剂： 无水乙醇、异丙醇。 实验前准备及重要注意事项 1．所有组分可在干燥、室温（15-30℃）环境稳定保存1年，将吸附柱置于2-8℃可保存更 长时间，加入RNase A的Buffer P1 置于2-8℃可稳定保存6个月。 2．第一次使用前，将RNase A溶液全部加入到Buffer P1 中，混匀，置于2-8℃保存。使用 前需在室温中放置一段时间，恢复至室温后使用。 3．第一次使用前应按照试剂瓶标签的说明先在Buffer PW中加入无水乙醇。 4．使用前请先检查Buffer P2和Buffer E3是否出现结晶或沉淀，如有结晶或沉淀现象， 可在37℃水浴几分钟，即可恢复澄清。 5．注意不要直接接触Buffer P2 和Buffer E3，使用后应立即盖紧盖子。 6．提取质粒的量和纯度与细菌培养浓度、菌株种类、质粒大小、质粒拷贝数等因素有关。 7．使用Buffer PS处理过的吸附柱最好立即使用，避免放置时间过长影响使用效果。 操作步骤 1．取15-50 ml过夜培养的新鲜菌液，加入离心管（自备）中，5000×g离心10分钟收集细菌， 尽量吸弃全部上清。 2．向留有菌体沉淀的离心管中加入2.5 ml Buffer P1（请先检查是否已加入RNase A）， 使用移液器或涡旋振荡器充分混匀，悬浮细菌沉淀。 注意：如果菌块未彻底混匀，将会影响裂解效果，使提取量和纯度偏低。 3．向离心管中加入2.5 ml Buffer P2，温和地上下颠倒混匀8-10次，使菌体充分裂解， 室温放置3-5分钟。此时溶液应变得清亮粘稠。 注意：温和混匀，不要剧烈震荡，以免打断基因组DNA，造成提取的质粒中混有基因组DNA片段。如 果溶液未变得清亮，提示可能菌量过大，裂解不彻底，应减少菌体量。 4．向离心管中加入2.5 ml Buffer E3，立即上下颠倒混匀8-10次，此时出现白色絮状沉淀。 注意：Buffer E3加入后应立即混匀，避免产生局部沉淀。 5．安装过滤器（Endo-Remover FX）的滤帽，将步骤4所得溶液转移至过滤器中，待白色 絮状沉淀浮于溶液上层，去掉过滤器的滤帽，对准干净的15 ml离心管（自备），慢慢推 动推柄（Plungers）过滤，使溶液尽可能多的通过，将滤液收集在离心管中。 6．向滤液中加入1/3溶液体积的异丙醇，上下颠倒混匀。 7．柱平衡：向已装入15 ml离心管的吸附柱（Spin Columns DX）中加入1 ml Buffer PS， 2500×g离心2分钟，倒掉离心管中的废液，将吸附柱重新放回离心管中。 8．将步骤6中滤液与异丙醇的混合溶液转移至已平衡的吸附柱（已装入收集管）中。 9．2500×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。 注意：吸附柱的最大容积为4 ml，所以第8步中所得溶液分2次过柱。 10.向吸附柱中加入2 ml Buffer PW（请先检查是否已加入无水乙醇），2500×g离心1分 钟，倒掉收集管中的废液。 11.重复步骤10。 12.将吸附柱重新放回收集管中，2500×g离心2分钟，倒掉废液，将吸附柱置于室温干燥 5分钟。 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.将吸附柱置于一个新的15 ml离心管中，向吸附膜的中间部位加入0.5-1 ml Endo-Free Buffer EB，室温放置2-5分钟，2500×g离心2分钟，将质粒溶液收集到离心管中。-20℃ 保存质粒。 注意：1）为了增加质粒的回收效率，可将得到的溶液重新加入到吸附柱中，室温放置2-5分钟，2500×g 离心2分钟，将质粒溶液收集到离心管中。 2）质粒拷贝数较低或>10 kb时，Endo-Free Buffer EB在65-70℃水浴预热，可以增加提取效率。
Product Introduction This kit is specially designed for the efficient and rapid extraction of plasmids from 15-50 ml of bacterial fluids. On the basis of cell lysis by alkaline lysis method, it adopts unique silicon matrix membrane adsorption technology to bind plasmid DNA efficiently and exclusively, and each adsorption column can adsorb up to 250 μg of plasmid DNA; at the same time, it adopts a special buffer system and endotoxin removal filter to effectively remove endotoxin, genomic DNA, RNA, protein and other impurities. The plasmids obtained from this kit are of high purity and stable quality, and can be used for cell transfection, as well as DNA sequencing, PCR, in vitro transcription, endonuclease digestion and other experiments. Self-contained reagents: anhydrous ethanol, isopropanol. Pre-experiment Preparation and Important Notes 1. All components are stable for 1 year in a dry, room temperature (15-30°C) environment, and longer by placing the adsorption columns at 2-8°C. Buffer P1 with RNase A is stable for 6 months at 2-8°C. 2. Before the first use, add all of the RNase A solution to Buffer P1, mix well, and store at 2-8°C. Before use, it needs to be left at room temperature for a period of time, return to room temperature and then use. 3. Anhydrous ethanol should be added to Buffer PW before the first use according to the instructions on the reagent bottle label. 4. Please check Buffer P2 and Buffer E3 for crystallization or precipitation before use. If there is any crystallization or precipitation, the clarification can be restored by taking a water bath at 37℃ for a few minutes. 5. Be careful not to touch Buffer P2 and Buffer E3 directly, and tighten the lid immediately after use. 6. The amount and purity of extracted plasmid is related to the concentration of bacterial culture, strain type, plasmid size, plasmid copy number and other factors. 7. The adsorption columns treated with Buffer PS should be used immediately to avoid leaving them for too long. Operation steps 1.Take 15-50 ml of fresh bacterial solution from the overnight culture, add it to a centrifuge tube (self-prepared) and centrifuge at 5000 × g for 10 minutes to collect the bacteria, and aspirate all the supernatant as much as possible. 2.Add 2.5 ml of Buffer P1 to the centrifuge tube in which the bacterial precipitate has been left (please check that RNase A has been added first) and suspend the bacterial precipitate by mixing thoroughly using a pipette or vortex shaker. Note: If the bacterial mass is not thoroughly mixed, it will affect the lysis effect and make the extraction amount and purity low. 3.Add 2.5 ml of Buffer P2 to the centrifuge tube, mix gently up and down 8-10 times to fully lyse the organisms, and leave at room temperature for 3-5 minutes. At this point the solution should become clear and viscous. Note: Mix gently, do not shake vigorously, so as not to interrupt the genomic DNA and cause genomic DNA fragments to be mixed in the extracted plasmid. If the solution does not become clear, it suggests that the amount of bacteria may be too large and the lysis is not complete, and the amount of bacteria should be reduced. 4.Add 2.5 ml of Buffer E3 to the centrifuge tube and mix immediately by turning up and down 8-10 times, at which time a white flocculent precipitate appears. Note: Buffer E3 should be mixed immediately after addition to avoid localized precipitation. 5.Install the cap of the filter (Endo-Remover FX), transfer the solution obtained in step 4 to the filter, wait until the white flocculent precipitate floats on the upper layer of the solution, remove the cap of the filter, align the filter with a clean 15 ml centrifuge tube (supplied), and slowly push the handle (Plungers) to filter, so that as much as possible of the solution passes through, and the filtrate is collected in the centrifuge tube. 6.Add 1/3 solution volume of isopropanol to the filtrate and mix upside down. 7.Column Equilibrium: Add 1ml Buffer PS to the adsorption column (Spin Columns DX) that has been loaded into a 15ml centrifuge tube, centrifuge for 2 minutes at 2500 x g. Pour off the waste liquid from the centrifuge tube and put the adsorption column back into the centrifuge tube. 8.The mixture of filtrate and isopropanol from step 6 was transferred to the equilibrated adsorption column (which had been loaded into a collection tube). 9.Centrifuge at 2500 x g for 1 minute, pour off the waste solution in the collection tube and put the adsorption column back into the collection tube. Note: The maximum volume of the adsorption column is 4 ml, so the solution obtained in step 8 is passed through the column in 2 times. 10.Add 2 ml of Buffer PW to the adsorption column (please check that anhydrous ethanol has been added first), centrifuge at 2500 × g for 1 min, and pour off the waste liquid in the collection tube. 11.Repeat step 10. 12.The adsorbent column was put back into the collection tube and centrifuged at 2500 × g for 2 min, the waste liquid was poured off, and the column was left to dry at room temperature for 5 min. 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. Place the adsorption column in a new 15 ml centrifuge tube, add 0.5-1 ml Endo-Free Buffer EB to the middle of the adsorbent membrane, leave it at room temperature for 2-5 minutes, centrifuge it at 2500 × g for 2 minutes, and collect the plasmid solution into the centrifuge tube. -20°C to store the plasmid. Note: 1) In order to increase the recovery efficiency of the plasmid, the obtained solution can be reintroduced into the adsorption column, left at room temperature for 2-5 minutes, centrifuged at 2500 x g for 2 minutes, and the plasmid solution can be collected into a centrifuge tube. 2) When the plasmid copy number is low or >10kb, Endo-Free Buffer EB can increase the extraction efficiency by preheating at 65-70°C in a water bath. |
安全和危险性（GHS）
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: