计算溶液所需的质量、体积或浓度。
为什么选择此级别
，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
室温。常规运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
产品内容：
产品简介：
内毒素是质粒提取中常见的污染物，由于真核细胞对内毒素非常敏感，因此，如 果质粒中含有内毒素会大大降低真核细胞转染效率。本试剂盒提供一种简单高效提取 无内毒素质粒的新方法，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；同时采用特殊的缓冲液系统和去内毒素buffer，有效去 除内毒素、基因组DNA、RNA、蛋白等杂质等。每次可处理100-300mL菌液，获得多 至2mg转染级质粒DNA，整个提取过程只需50分钟。本试剂盒所得质粒纯度高、提取 量大，特别适用于细胞转染，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。
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.Buffer P1在使用前先加入RNase A（将试剂盒中提供的RNase A 全部加入），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在Buffer PW中加入无水乙醇。
4.使用前请先检查Buffer P2、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Buffer P2和Buffer E3含有刺激性物质，请戴手套操作，使用后应立即盖紧盖子。
6.使用Buffer PS处理过的吸附柱放置15-30分钟后再进行混合液过柱，效果较好，不 建议放置超过30分钟使用。
7.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。
8.质粒提取菌液量应不超过表1和表2中推荐的体积，高拷贝质粒提取的P1、P2和E3 溶液的用量均为12 mL；而对于低拷贝质粒提取，P1、P2和E3溶液的用量应根据 菌液量调整P1、P2和E3溶液的用量，可参考表3：
注意：ODV=OD600×V，V为菌液量（mL）；推荐OD600 为1-4。
9. 低拷贝质粒建议使用裂解液体积，详见下表：
注意：以OD600=4时为例。
操作步骤：
|
内毒素是质粒提取中常见的污染物，由于真核细胞对内毒素非常敏感，因此，如 果质粒中含有内毒素会大大降低真核细胞转染效率。本试剂盒提供一种简单高效提取 无内毒素质粒的新方法，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；同时采用特殊的缓冲液系统和去内毒素buffer，有效去 除内毒素、基因组DNA、RNA、蛋白等杂质等。每次可处理100-300mL菌液，获得多 至2mg转染级质粒DNA，整个提取过程只需50分钟。本试剂盒所得质粒纯度高、提取 量大，特别适用于细胞转染，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。
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.Buffer P1在使用前先加入RNase A（将试剂盒中提供的RNase A 全部加入），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在Buffer PW中加入无水乙醇。
4.使用前请先检查Buffer P2、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Buffer P2和Buffer E3含有刺激性物质，请戴手套操作，使用后应立即盖紧盖子。
6.使用Buffer PS处理过的吸附柱放置15-30分钟后再进行混合液过柱，效果较好，不 建议放置超过30分钟使用。
7.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。
8.质粒提取菌液量应不超过表1和表2中推荐的体积，高拷贝质粒提取的P1、P2和E3 溶液的用量均为12 mL；而对于低拷贝质粒提取，P1、P2和E3溶液的用量应根据 菌液量调整P1、P2和E3溶液的用量，可参考表3：
注意：ODV=OD600×V，V为菌液量（mL）；推荐OD600 为1-4。
9. 低拷贝质粒建议使用裂解液体积，详见下表：
注意：以OD600=4时为例。
操作步骤：
1. 取150 mL过夜培养的菌液，加入离心管（自备）中，12,000×g离心2-3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12 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。对于低拷贝质粒，作用菌液
量较大时需要按比例增加P1、P2、E3的使用量，请参考表3。
3. 向离心管中加入12 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. 此时进行柱平衡：向已装入收集管中的吸附柱（Spin Columns DZ）中加入2 mL Buffer PS，12,000 ×g离心2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：第4步柱平衡完成后（Buffer PS过柱）直至用于第7步（上清与异丙醇混合液过柱）建议有
15-30分钟的时间差，可使提取得率提高。
5. 向离心管中加入12 mL Buffer E3，立即上下颠倒混匀8-10次，此时出现白色絮状沉淀，室温放置5分钟。12,000×g离心10分钟，将上清转移至干净离心管（自备） 中，注意不要带入沉淀。
注意：Buffer E3 加入后应立即混匀，避免产生局部沉淀。
6. 加入0.3倍上清体积的异丙醇，上下颠倒混匀。
注意：加入异丙醇过多容易导致RNA污染。
7. 将上清与异丙醇的混合溶液转移到步骤4已平衡好的吸附柱DZ（已装入收集管） 中。12,000×g离心2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15 mL，所以第7步中所得溶液分多次过柱。如果离心机转子倾角较大时，建议加入吸附柱的溶液体积不超过10 mL，以防发生漏液现象。
8.向吸附柱中加入10 mL Buffer PW（请先检查是否已加入无水乙醇），12,000×g离 心2分钟，倒掉收集管中的废液，将吸附柱重新放回收集管中。
9.重复步骤8。
10.向吸附柱中加入10 mL Endo-Free Buffer PW，12,000×g离心2分钟，倒掉收集管 中的废液，将吸附柱重新放回收集管中。
11.将吸附柱重新放回收集管中，12,000×g离心5分钟，倒掉废液，将吸附柱置于室温 数分钟，以彻底晾干吸附柱中残余的漂洗液。
注意：这一步的目的是将吸附柱中残余乙醇去除，乙醇的残留会影响后续的酶促反应（酶切、PCR 等）。
12. 将吸附柱置于一个新的离心管中，向吸附膜的中间部位加入1-3mLEndo-Free Buffer EB，室温放置2-5分钟，12,000×g离心5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， 12,000×g离心5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 content:
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Product Introduction:
Endotoxins are a common pollutant in plasmid extraction. Due to the high sensitivity of eukaryotic cells to endotoxins, the presence of endotoxins in plasmids can greatly reduce the transfection efficiency of eukaryotic cells. This kit provides a simple and efficient new method for extracting endotoxin free plasmids. Based on the alkaline lysis method for cell lysis, a unique silicon matrix membrane adsorption technology is used to efficiently and specifically bind plasmid DNA; At the same time, a special buffer system and endotoxin removal buffer are used to effectively remove impurities such as endotoxins, genomic DNA, RNA, proteins, etc. Each time, 100-300mL of bacterial solution can be processed to obtain up to 2mg of transfected plasmid DNA, and the entire extraction process only takes 50 minutes. The plasmid obtained from this kit has high purity and extraction capacity, making it particularly suitable for cell transfection. It can also be used for DNA sequencing, PCR, in vitro transcription, endonuclease digestion and other experiments.
Self prepared reagents: anhydrous ethanol, isopropanol.
Notes:
1. All components can be stably stored for 1 year in a dry, room temperature (15-30 ℃) environment. The adsorption column can be stored for a longer time at 2-8 ℃.
2.Buffer P1 with RNase A added can be stably stored for 6 months at 2-8 ℃. Before use, add RNase A to Buffer P1 (add all RNase A provided in the reagent kit), mix well, and store at 2-8 ℃. Before use, it is necessary to leave it at room temperature for a period of time, and then use it after returning to room temperature.
3.Before the first use, anhydrous ethanol should be added to the Buffer PW according to the instructions on the reagent bottle label.
4. Before use, please check if there is any crystallization or precipitation in Buffer P2 and Buffer E3. If there is any crystallization or precipitation, you can take a water bath at 37 ℃ for a few minutes to restore clarity.
5. Note that Buffer P2 and Buffer E3 contain irritating substances. Please wear gloves when operating and immediately cover the lid after use.
6. The adsorption column treated with Buffer PS should be left for 15-30 minutes before passing the mixed solution through the column. The effect is good, and it is not recommended to leave it for more than 30 minutes.
7.The amount and purity of plasmid extraction are related to factors such as bacterial culture concentration, strain type, plasmid size, and plasmid copy number.
8. The amount of plasmid extracted bacterial solution should not exceed the recommended volume in Tables 1 and 2. The amount of P1, P2, and E3 solutions extracted from high copy plasmids is 12 mL each; For the extraction of low copy plasmids, the dosage of P1, P2, and E3 solutions should be adjusted according to the amount of bacterial solution, as shown in Table 3:
Note: ODV=OD600 × V, where V is the amount of bacterial solution (mL); The recommended OD600 is 1-4.
9. It is recommended to use the volume of lysis solution for low copy plasmids, as shown in the table below:
Note: Taking OD600=4 as an example.
Operation steps:
1. Take 150 mL of overnight cultured bacterial solution and add it to a centrifuge tube (provided by oneself). Centrifuge 12000 × g for 2-3 minutes to collect bacteria, and try to discard all the supernatant as much as possible.
2. Add 12 mL of Buffer P1 (please check if RNase A has been added first) to the centrifuge tube containing bacterial precipitation. Mix thoroughly with a pipette or vortex oscillator to suspend bacterial precipitation.
Attention: If the bacterial blocks are not thoroughly mixed, it will affect the cracking effect, resulting in low extraction amount and purity. For low copy plasmids, the acting bacterial solution
When the quantity is large, it is necessary to increase the usage of P1, P2, and E3 proportionally, please refer to Table 3.
3. Add 12 mL of Buffer P2 to the centrifuge tube, gently invert and mix 8-10 times, allowing the bacterial cells to fully lyse. Let it stand at room temperature for 5 minutes. At this point, the solution should become clear and viscous.
Attention: Mix gently and do not shake vigorously to avoid interrupting genomic DNA and mixing genomic DNA fragments in the extracted plasmid.
If the solution does not become clear, it indicates that the bacterial count may be too high and the lysis may not be complete. The bacterial count should be reduced.
4. At this point, perform column equilibrium: add 2 mL of Buffer PS to the Spin Columns DZ that has been loaded into the collection tube, centrifuge at 12000 × g for 2 minutes, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. Note: After the completion of column equilibrium in step 4 (Bu ff er PS passing through the column), it is recommended to use it for step 7 (supernatant and isopropanol mixture passing through the column)
A time difference of 15-30 minutes can increase the extraction yield.
5. Add 12 mL of Buffer E3 to the centrifuge tube, immediately invert and mix 8-10 times. At this point, white flocculent precipitates appear, and let it stand at room temperature for 5 minutes. Centrifuge 12000 × g for 10 minutes and transfer the supernatant to a clean centrifuge tube (provided), taking care not to introduce sediment.
Attention: Bu ff er E3 should be mixed immediately after addition to avoid local precipitation.
6. Add 0.3 times the volume of the supernatant with isopropanol, invert and mix well.
Attention: Adding too much isopropanol can easily lead to RNA contamination.
7. Transfer the mixed solution of the supernatant and isopropanol to the equilibrium adsorption column DZ (loaded into the collection tube) in step 4. Centrifuge 12000 × g for 2 minutes, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. Note: The maximum volume of the adsorption column is 15 mL, so the solution obtained in step 7 is passed through the column multiple times. If the inclination angle of the centrifuge rotor is large, it is recommended to add a solution volume of no more than 10 mL to the adsorption column to prevent liquid leakage.
8. Add 10 mL of Buffer PW to the adsorption column (please check if anhydrous ethanol has been added first), centrifuge at 12000 × g for 2 minutes, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.
9. Repeat step 8.
10. Add 10 mL Endo Free Buffer PW to the adsorption column, centrifuge at 12000 × g for 2 minutes, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.
11. Place the adsorption column back into the collection pipe, centrifuge at 12000 × g for 5 minutes, discard the waste liquid, and place the adsorption column at room temperature for a few minutes to thoroughly dry the residual rinsing solution in the column.
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.).
12. Place the adsorption column in a new centrifuge tube, add 1-3mLEndo Free Buffer EB to the middle of the adsorption membrane, let it stand at room temperature for 2-5 minutes, centrifuge 12000 × g for 5 minutes, and collect the plasmid solution into the centrifuge tube- Store the plasmid at 20 ℃.
Attention:1） To increase the recovery efficiency of plasmids, the obtained solution can be added back to the adsorption column, left at room temperature for 2-5 minutes, centrifuged at 12000 × g for 5 minutes, and collected into a centrifuge tube.
2) When the plasmid copy number is low or>10 kb, preheating the Endo Free Buffer EB in a water bath at 65-70 ℃ can increase the extraction efficiency.
规格
英文名称
GoldHi EndoFree Plasmid Maxi Kit
储存与运输
储存条件
室温
运输条件
常规运输
技术文档
高级数据
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:
技术文档和文章
溶液计算器
产品仅提供给经过验证的企业、机构和合格的专业人员用于研发。不适用于人类、动物、诊断或治疗。
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