计算溶液所需的质量、体积或浓度。
无内毒素高纯度质粒大量快速提取试剂盒
有货
为什么选择此级别
BioReagent,分子生物学级 ，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
本试剂盒采用独特的硅胶膜吸附技术，高效专一地结合质粒DNA，同时采用特殊的溶液P4和过滤器CS1，可有效地去除内毒素和蛋白等杂质，整个提取过程方便快捷， 仅需1h。使用本试剂盒提取的质粒DNA可适用于各种常规实验，包括酶切、PCR、 测序、连接、转化和细胞转染等。
推荐每次培养基使用量：高拷贝质粒推荐使用量为100 mL，得率一般在500-1500 μg左右；低拷贝质粒推荐使用量为200 mL，得率一般在200-600 μg左右。
产品特点
1. 不需要使用有毒的苯酚、氯仿等试剂，也不需要乙醇沉淀。从100~200 mL大肠杆菌LB培养液中，可快速提取0.2~0.4 mg高质量的高拷贝质粒，提取率达80~90%。
2. 获得的质粒产量高，超螺旋比例高，纯度好，可直接用于酶切、转化、PCR、体外转录、测序和转染等各种分子生物学实验。
本试剂盒在室温储存18个月不影响使用效果。
注意事项
1. 溶液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。
2. 使用前先检查溶液P2和P4是否出现结晶或者沉淀，如有结晶或者沉淀现象，可在37℃水浴中加热几分钟，即可恢复澄清。
3. 注意不要直接接触溶液P2和P4，使用后应立即盖紧盖子。
4. 使用过滤器时请将推柄小心缓慢地从过滤管中抽出，避免滤膜因压力而松动。
5. 提取的质粒量与细菌培养浓度、质粒拷贝数等因素有关。如果所提质粒为低拷贝质粒或大于10kb的大质粒，应加大菌体使用量，同时按比例增加P1、P2、P4的用量；洗脱缓冲液推荐在65-70℃水浴中预热。可以适当延长吸附和洗脱时间，以提高提取效率。
6. 本试剂盒仅限科研使用，不可用于临床诊断及其他用途。
自备试剂：无水乙醇
操作步骤
注意：
- 第一次使用前请先在漂洗液WB中加入100 mL无水乙醇，充分混匀，加入后请及时标记已加入乙醇，以免多次加入！
- 将RNase A（干粉）倒入溶液P1中并使用P1冲洗离心管，混匀，每次使用后置于2-8℃保存。
1. 柱平衡步骤：向吸附柱CP6中（吸附柱放入50 mL收集管中）加入2.5 mL的平衡液BL，8,000 rpm (~8,228×g) 离心2 min，倒掉收集管中的废液，将吸附柱重新放回收集管中。（用平衡液处理过的柱子最好立即使用）。
2. 取100 mL（根据培养菌体的浓度选择合适的量，低拷贝推荐用200 mL）过夜培养的菌液加入离心管，室温8,000 rpm (~8,228×g) 离心3 min收集细菌，尽量吸除上清。为确保上清液全部吸取，可用干净的吸水纸吸去瓶壁上的水滴。
注意：菌液较多时可以通过多次离心将菌体沉淀收集到一个离心管中，菌液量以能够充分裂解为佳，菌液过多会导致裂解不充分从而降低质粒的提取效率。
3. 向留有菌体沉淀的离心管中加入8 mL溶液P1（请先检查是否已加入RNase A），使用移液器或涡旋振荡器彻底悬浮细菌细胞沉淀。
注意：请务必彻底悬浮细菌沉淀，如果有未彻底混匀的菌块，会影响裂解效果，导致提取量和纯度偏低。对于低拷贝质粒，加大菌体用量的同时按比例增加P1、P2和P4 的用量。
4. 向离心管中加入8 mL溶液P2，立即温和地上下翻转6-8次，使菌体充分裂解，室温放置5 min。
注意：温和地混匀，不要剧烈震荡，以免污染基因组DNA。此时菌液应变得清亮粘稠，如果未变得清亮，可能由于菌体过多，裂解不彻底，应减少菌体量。
5. 向离心管中加入8 mL溶液P4，立即温和地上下翻转6-8次，充分混匀，至溶液出现白色分散絮状沉淀。然后室温放置10 min左右。8,000 rpm (~8,228×g) 离心5-10 min，使白色沉淀离至管底（可适当增加离心时间），将全部溶液小心倒入过滤器CS1中（请避免倒入大量沉淀而阻塞过滤器），慢慢推动推柄过滤，滤液收集在干净的50 mL的管中（自备）。
注意：加入溶液P4后应立即混匀，避免产生局部沉淀。如果离心后倒入过滤器CS1中的溶液有白色沉淀也不会影响过滤。如果菌体过多（>100 mL），推荐延长离心时间至20-30 min。
6. 向滤液中加入0.3倍滤液体积的异丙醇（加入异丙醇过多容易导致RNA污染），上下颠倒 混匀后转移到吸附柱CP6中（吸附柱放入50 mL收集管中）。
注意：过滤后滤液会损失，根据损失的不同请加入不同体积的异丙醇。吸附柱CP6的最大容积为15 mL，所以需要分两次过柱。个别情况下离心机转子倾角较大，此时，建议加入吸附柱CP6的溶液体积不超过10 mL，以防产生漏液现象。
7. 室温8,000 rpm (~8,228×g) 离心2 min，倒掉收集管中的废液，将吸附柱CP6重新放回收集管中。
注意：将第6步中所得溶液分两次过柱，每次均按以上条件操作。
8. 向吸附柱CP6中加入8 mL去蛋白液PD，8,000 rpm (~8,228×g) 离心2 min，弃掉收集管中的废液，将吸附柱重新放回收集管中。
9. 向吸附柱CP6中加入10 mL漂洗液WB（请先检查是否已加入无水乙醇），8,000 rpm (~8,228×g) 离心2 min，弃掉收集管中的废液，将吸附柱重新放回收集管中。
10. 重复操作步骤9。
11. 向吸附柱CP6中加入3 mL无水乙醇，室温8,000 rpm (~8,228×g) 离心2 min，倒掉废液。
12. 将吸附柱CP6重新放回收集管中，8,000 rpm (~8,228×g) 离心5 min，目的是将吸附柱中残余的乙醇去除。
注意：乙醇的残留会影响后续的酶促反应（酶切、PCR等）实验。为确保下游实验不受残留乙醇的影响，建议将吸附柱CP6开盖，置于室温放置数分钟，以彻底晾干吸附材料中残余的乙醇。
13. 将吸附柱CP6置于一个干净的50 mL收集管中，向吸附膜的中间部位悬空滴加1-2 mL洗脱缓冲液EB，室温放置5 min，然后室温8,000 rpm (~8,228×g) 离心2 min。将50 mL收集管中的洗脱液全部移入一个干净的1.5 mL离心管，-20℃保存。
注意：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，重复步骤11。洗脱液的pH值对于洗脱效率有很大影响。若用ddH₂O做洗脱液应保证其pH值在 7.5-8.0 范围内，pH值低于7.0会降低洗脱效率。洗脱缓冲液用量的多少主要是依据质粒的拷贝数以及实验所需要的浓度来确定。洗脱缓冲液体积不少于1 mL，体积过小影响回收效率。DNA产物应保存在-20℃，以防DNA降解。
可选步骤（如果需要更高浓度的质粒，可进行如下操作）：
14. 每1 mL洗脱液加入1.42 mL异丙醇以及0.42 mL 5M NaCl（客户自备），混匀，室温放置5 min，8,000 rpm (~8,228×g) 离心10 min，小心弃上清。
15. 加入0.5 mL的70%乙醇洗涤沉淀, 室温8,000 rpm (~8,228×g) 离心5 min，小心弃乙醇。
16. 重复操作步骤15。
17. 空气中干燥DNA沉淀5-10 min，根据需要用适当体积的EB缓冲液溶解沉淀。
This kit utilizes a unique silica membrane adsorption technology for efficient and specific binding of plasmid DNA. It also employs a special Solution P4 and Filter CS1 to effectively remove impurities such as endotoxins and proteins. The entire extraction process is convenient and rapid, taking only 1 hour. Plasmid DNA extracted with this kit is suitable for various routine experiments, including restriction enzyme digestion, PCR, sequencing, ligation, transformation, and cell transfection.
Recommended Culture Volume per Use: For high-copy plasmids, the recommended culture volume is 100 mL, typically yielding 500–1500 μg. For low-copy plasmids, the recommended culture volume is 200 mL, typically yielding 200–600 μg.
Product Features
No need for toxic reagents like phenol or chloroform, nor for ethanol precipitation. From 100–200 mL of E. coli LB culture, 0.2–0.4 mg of high-quality high-copy plasmid can be rapidly extracted with an extraction efficiency of 80–90%.
The obtained plasmid DNA is characterized by high yield, a high proportion of supercoiled form, and high purity. It can be used directly in various molecular biology experiments such as restriction enzyme digestion, transformation, PCR, in vitro transcription, sequencing, and transfection.
This kit can be stored at room temperature for 18 months without affecting performance.
Precautions
Before using Solution P1, add RNase A (add the entire vial provided in the kit), mix well, and store at 2–8°C.
Before use, check Solutions P2 and P4 for crystallization or precipitation. If present, warm the solutions in a 37°C water bath for a few minutes to restore clarity.
Avoid direct contact with Solutions P2 and P4. Tightly cap the bottles after use.
When using the filter, carefully and slowly withdraw the plunger from the filter housing to avoid loosening the membrane due to pressure changes.
The amount of plasmid extracted depends on factors such as bacterial culture concentration and plasmid copy number. For low-copy plasmids or large plasmids (>10 kb), increase the amount of bacterial culture and proportionally increase the volumes of Solutions P1, P2, and P4. Pre-heating the Elution Buffer EB in a 65–70°C water bath is recommended. Adsorption and elution times can be appropriately extended to improve extraction efficiency.
For research use only. Not for clinical diagnosis or other uses.
Materials Required but Not Provided
Anhydrous ethanol
Procedure
Notes:
Before first use, add 100 mL of anhydrous ethanol to Wash Buffer WB. Mix thoroughly. Mark the bottle immediately after adding ethanol to avoid double addition.
Pour the RNase A (powder) into Solution P1, using P1 to rinse the vial. Mix well. Store at 2–8°C after each use.
Column Equilibration Step: Add 2.5 mL of Equilibration Buffer BL to a Spin Column CP6 (placed in a 50 mL Collection Tube). Centrifuge at 8,000 rpm (~8,228×g) for 2 minutes. Discard the flow-through and place the column back into the collection tube. (Columns processed with Equilibration Buffer BL are best used immediately).
Transfer the overnight bacterial culture (use 100 mL for high-copy plasmids; for low-copy plasmids, 200 mL is recommended; adjust based on culture concentration) to a centrifuge tube. Centrifuge at room temperature at 8,000 rpm (~8,228×g) for 3 minutes to collect the bacterial pellet. Remove the supernatant as completely as possible. To ensure complete removal, absorb any residual droplets on the tube wall with clean absorbent paper.
Note: If processing a large volume of culture, the bacterial pellets from multiple centrifuge runs can be combined into one tube. The optimal bacterial amount allows for complete lysis; excessive bacteria can lead to insufficient lysis and reduced plasmid extraction efficiency.
Add 8 mL of Solution P1 (ensure RNase A has been added) to the centrifuge tube containing the bacterial pellet. Resuspend the bacterial pellet thoroughly using a pipette or a vortex mixer.
*Note: Ensure the bacterial pellet is completely resuspended. Any unhomogenized clumps can affect lysis efficiency, resulting in lower yield and purity. For low-copy plasmids, when increasing the bacterial culture volume, proportionally increase the volumes of Solutions P1, P2, and P4.*
Add 8 mL of Solution P2 to the centrifuge tube. Immediately and gently invert the tube 6–8 times to lyse the cells thoroughly. Allow to stand at room temperature for 5 minutes.
Note: Mix gently. Avoid vigorous shaking to prevent genomic DNA contamination. The suspension should become clear and viscous. If it does not, it may be due to excessive bacterial quantity leading to incomplete lysis; reduce the amount of starting culture.
Add 8 mL of Solution P4 to the centrifuge tube. Immediately and gently invert the tube 6–8 times until a white, dispersed, flocculent precipitate appears. Allow to stand at room temperature for approximately 10 minutes. Centrifuge at 8,000 rpm (~8,228×g) for 5–10 minutes to sediment the white precipitate to the bottom of the tube (centrifugation time can be extended if necessary). Carefully pour the entire supernatant into Filter CS1 (avoid pouring in large amounts of precipitate that could clog the filter). Slowly push the plunger to filter. Collect the filtrate in a clean 50 mL tube (user-provided).
Note: Mix immediately after adding Solution P4 to avoid localized precipitation. If the solution poured into Filter CS1 after centrifugation contains some white precipitate, filtration is still possible. If excessive bacterial culture (>100 mL) was used, extending the centrifugation time to 20–30 minutes is recommended.
Add 0.3 volumes of isopropanol relative to the filtrate volume to the filtrate (excess isopropanol may lead to RNA contamination). Mix thoroughly by inverting. Transfer the mixture to the Spin Column CP6 (placed in a 50 mL Collection Tube).
Note: Filtrate volume may vary after filtration; adjust the volume of isopropanol added accordingly. The maximum capacity of Spin Column CP6 is 15 mL, so the sample needs to be loaded in two separate centrifugations. In some cases, depending on the rotor angle, it is recommended not to exceed 10 mL per loading to prevent leakage.
Centrifuge at room temperature at 8,000 rpm (~8,228×g) for 2 minutes. Discard the flow-through and place the Spin Column CP6 back into the collection tube.
Note: Load the solution from step 6 in two batches, centrifuging each batch under the conditions described above.
Add 8 mL of Protein Removal Solution PD to the Spin Column CP6. Centrifuge at 8,000 rpm (~8,228×g) for 2 minutes. Discard the flow-through and return the column to the collection tube.
Add 10 mL of Wash Buffer WB (ensure anhydrous ethanol has been added) to the Spin Column CP6. Centrifuge at 8,000 rpm (~8,228×g) for 2 minutes. Discard the flow-through and return the column to the collection tube.
Repeat step 9.
Add 3 mL of anhydrous ethanol to the Spin Column CP6. Centrifuge at room temperature at 8,000 rpm (~8,228×g) for 2 minutes. Discard the flow-through.
Place the Spin Column CP6 back into the collection tube. Centrifuge at 8,000 rpm (~8,228×g) for 5 minutes to remove residual ethanol from the column.
*Note: Residual ethanol can affect downstream enzymatic reactions (e.g., restriction digestion, PCR). To ensure downstream experiments are not affected, it is recommended to open the cap of the Spin Column CP6 and allow it to stand at room temperature for several minutes to thoroughly air-dry any remaining ethanol.*
Place the Spin Column CP6 into a clean 50 mL collection tube. Carefully add 1–2 mL of Elution Buffer EB to the center of the silica membrane, ensuring the pipette tip does not touch the membrane. Allow to stand at room temperature for 5 minutes. Centrifuge at room temperature at 8,000 rpm (~8,228×g) for 2 minutes. Transfer the entire eluate from the 50 mL collection tube to a clean 1.5 mL centrifuge tube. Store at –20°C.
Note: To maximize plasmid recovery, the eluted solution can be reapplied to the column and the centrifugation step repeated. The pH of the elution buffer significantly affects elution efficiency. If using ddH₂O for elution, ensure its pH is within the range of 7.5–8.0; pH values below 7.0 reduce elution efficiency. The volume of Elution Buffer EB used depends on the plasmid copy number and the desired concentration. Use at least 1 mL of elution buffer; smaller volumes reduce recovery efficiency. DNA products should be stored at –20°C to prevent degradation.
Optional Step (To obtain higher concentration plasmid, the following procedure can be performed):
For every 1 mL of eluate, add 1.42 mL of isopropanol and 0.42 mL of 5 M NaCl (user-provided). Mix thoroughly and stand at room temperature for 5 minutes. Centrifuge at 8,000 rpm (~8,228×g) for 10 minutes. Carefully discard the supernatant.
Add 0.5 mL of 70% ethanol to wash the precipitate. Centrifuge at room temperature at 8,000 rpm (~8,228×g) for 5 minutes. Carefully discard the ethanol.
16. Repeat step 15.
Air-dry the DNA precipitate for 5–10 minutes. Dissolve the precipitate in an appropriate volume of Elution Buffer EB as needed.
规格
别名
无内毒高纯度快速质粒大提试剂盒 | 无内毒高纯度质粒DNA大量抽提试剂盒（离心柱法）
英文别名
Endo-Free High Pure Plasmid Maxi Rapid Extraction Kit | Endo-Free High Pure Rapid Maxi Plasmid Kit
规格或纯度
BioReagent, 分子生物学级
英文名称
Endo-Free High Purity Rapid Plasmid Maxi Kit
应用
DNA Extraction
样品类型
质粒
储存与运输
储存条件
室温
运输条件
常规运输
稳定性与储存
Store at room temperature long term (18 months).
组分与储存
| E1521037 | Component | 10T | Storage |
| E1521037A | 平衡液BL | 30 mL | RT. |
| E1521037B | RNase A（干粉） | 1 EA | RT. |
| E1521037C | 溶液P1 | 100 mL | RT. |
| E1521037D | 溶液P2 | 100 mL | RT. |
| E1521037E | 溶液P4 | 100 mL | RT. |
| E1521037F | 去蛋白液PD | 100 mL | RT. |
| E1521037G | 漂洗液WB | 25 mL×2 | RT. |
| E1521037H | 洗脱缓冲液EB | 30 mL | RT. |
| E1521037I | 过滤器CS1 | 1 EA×10 | RT. |
| E1521037J | 吸附柱CP6 | 1 EA×10 | RT. |
| E1521037K | 收集管（50 mL） | 1 EA×20 | RT. |
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:
通过匹配包装上的批号来查找并下载产品的 COA，每批产品都进行了严格的验证，您可放心使用！
| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0535151 |
分析证书 | E1521037 |
技术文档和文章
溶液计算器
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