本试剂盒适用于从50~200 mL细菌培养液中提取去内毒素质粒DNA，该方法采用超顺磁性的磁性粒子纯化技术及去内毒素结合液去除内毒素技术，提取过程中无需使用有毒的酚氯仿抽提，操作简单方便，纯化的质粒可直接用于酶切、PCR、一代测序和细胞转染等相关实验。其处理后的产物用于科研检测使用。
| M1521549 | 组件名称
| 10T | 50T | 储存温度
|
| M1521549A | RNase A
| 40 mg | 200 mg | -20℃.
|
| M1521549B | 磁珠MXVB
| 3.3 mL | 16.5 mL | 2-8℃.
|
| M1521549C | 内毒素结合液ERB
| 154 mL | 770 mL | RT.
|
| M1521549D | 内毒素去除液ETR
| 30 mL | 150 mL | RT.
|
| M1521549E | 重悬液S1
| 112 mL | 560 mL | RT.
|
| M1521549F | 裂解液S2
| 112 mL | 560 mL
| RT.
|
| M1521549G | 沉淀液N5
| 55 mL | 275 mL | RT.
|
| M1521549H | 洗涤液BW1
| 100 mL | 495 mL | RT.
|
| M1521549I | 洗涤液DW2
| 200 mL | 495 mL×2
| RT.
|
| M1521549J | 洗脱液DE
| 33 mL | 165 mL | RT. |
M1521549A：-20℃保存，12个月。
M1521549B：2-8℃保存，12个月。
M1521549C, M1521549D, M1521549E, M1521549F, M1521549G, M1521549H, M1521549I, M1521549J：室温保存，12个月。
适配仪器
手工提取
注意事项
1. 使用前请仔细阅读本说明书。
2. 本试剂盒中的多种缓冲液含有刺激性的胍盐，务必戴上手套，并按照安全标准预防措施来处理。不要让缓冲液接触到皮肤、眼睛以及黏膜，如果确实发生，请立即用大量清水清洗并就医。
3. 如因气温较低，裂解液S2出现沉淀，可37℃水浴至沉淀完全溶解后使用。
4. 如因气温较低，用力振荡后磁珠无法重悬，请勿使用。
5. 本试剂盒中的多种缓冲液含胍盐，请勿用氧化性消毒剂如次氯酸钠进行处理，否则会释放有毒气体，须按医疗废物进行处理。
6. 本试剂盒需配套使用，不同批号试剂盒中各组分不可以互换或混用。
使用方法
1. 样本要求
- 适用标本类型：50~200 mL细菌培养液。
- 标本保存和运输：标本可立即用于测试，也可以保存于-70℃或更低温度待测，保存期为6个月，应避免反复冻融。标本运输采用冷链运输。
- 冻融要求：速冻速融，避免反复冻融。
2. 自备试验器材
3. 试剂准备
4. 操作步骤
磁分离操作建议（步骤4.10开始）：离心管置于磁力架后，轻轻地左右旋转，待磁珠聚集于贴近磁力架的管壁后，轻轻地颠倒磁力架数次，使管盖上的磁珠也聚集到管壁，静置数分钟至溶液澄清即可（静置时长视磁力架磁性而定）。
4.1 在培养瓶中加入50~200 mL含抗生素的LB培养液，将含目的载体的菌种接种至培养瓶中，37℃摇床培养12~16小时。
注意：建议使用LB培养基，如使用TB、YT等丰富培养基来培养细菌，样本量需减半。
4.2 使用对应离心管10,000×g离心1分钟收集菌体，大体积如200 mL菌液可用50 mL离心管分四次离心收集。倒弃培养基，在吸水纸上轻轻拍打吸尽残液。
4.3 加入10 mL S1/RNase A混合液，高速涡旋重悬细菌。
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。
4.4 往重悬液中加入10 mL裂解液S2，轻柔颠倒混匀颠倒混匀2~3分钟。室温放置8~10分钟，期间每2分钟颠倒混匀20~30次。
注意：需轻轻颠倒混匀，涡旋会引起基因组DNA污染，溶液变得粘稠而透亮表明细菌已充分裂解。若出现裂解液S2裂解不完全的情况，建议适当减少菌量。
4.5 随即加入5 mL沉淀液N5，立即颠倒混匀15~25次让溶液彻底中和。
注意：加入沉淀液N5后应立即颠倒混匀，以防止沉淀团聚而影响中和效果，菌液用量较多时，中和也会较难进行，中和时需要稍用力振荡数次至溶液彻底中和，中和后沉淀应为白色，如未彻底中和可增加混匀次数至完全中和。
4.6 10,000×g离心5分钟。
4.7 可按需求选择以下操作步骤：
①（快速提取，适用于普通转染级质粒提取，提取质粒内毒素含量约200~500 EU/μg）
无需处理，按照步骤4.8进行后续操作。
②（增强型去内毒素质粒提取，适用于需求更低内毒素含量的质粒提取，提取质粒内毒素含量约0.1~2.5 EU/μg）
转移上清至新的离心管中，加入1/10体积的内毒素去除液ETR，如600 µL上清加入60 µL内毒素去除液ETR。
颠倒混匀后置4℃冰箱冷藏10分钟，取出后42℃温浴10分钟。10,000×g离心3分钟。
4.8 转移全部上清液至新的50 mL离心管中。
4.9 加入上清液0.7倍体积去内毒素结合液ERB，300 µL磁珠MXVB，涡旋混匀5~7分钟使磁珠吸附DNA。
4.10 转移至磁力架上进行磁分离至磁吸完全，吸弃溶液，避免吸到磁珠。
4.11 加入9 mL洗涤液BW1，静置浸泡3~5分钟，取出颠倒混匀30秒后放至50 mL磁力架上进行磁分离至溶液澄清。吸弃溶液，避免吸到磁珠。（静置浸泡可使产量更稳定，此步骤较关键，不能省略）
4.12 加入9 mL洗涤液DW2，涡旋30秒打散磁珠后转50 mL磁力架上进行磁分离至溶液澄清。吸弃溶液，避免吸到磁珠。
4.13 再加入3 mL洗涤液DW2，涡旋30秒打散磁珠后转至50 mL磁力架上进行磁分离至溶液澄清。吸弃溶液，避免吸到磁珠。
4.14 将离心管置磁力架上进行磁分离，吸弃管内及管盖所有溶液，空气干燥5~10分钟。
注意：乙醇残留会抑制后续的酶反应，所以晾干时要确保乙醇挥发干净。干燥过程中如有液体回流到管底，可使用移液器吸弃，至磁珠表面无明显液体状即可，应避免使用烘箱等加热干燥，过度干燥会影响DNA洗脱，导致DNA得率下降。
4.15 加入1-3 mL洗脱液DE，随后使用移液器吸取管中溶液冲洗管壁，确保管壁上的残留磁珠被彻底冲洗并聚集到管底部。
4.16 将洗脱溶液及磁珠转移至1-2个 2.0 mL离心管中，涡旋混匀30秒左右。
4.17 将离心管置于60℃温浴5分钟，然后高速涡旋30秒。
4.18 瞬时离心收集管盖液滴至管中，转移至磁力架上进行磁分离。
4.19 把DNA溶液转移至新的离心管中待用。如果不立即使用，请存储于-15~-25℃，长期保存请放置于-70℃或更低的温度。
方法的局限性
1. 对于某些取样不佳的样本，提取产物中可能存在少量杂质抑制PCR扩增。
2. 样本核酸的提取效果与样本收集、处理、运输和保存质量密切相关，其中任何失误都将会导致结果不准确。
This kit is designed for the extraction of endotoxin-free plasmid DNA from 50–200 mL of bacterial culture. The method utilizes superparamagnetic particle purification technology combined with an endotoxin removal buffer system to eliminate endotoxins. The extraction process does not require the use of toxic phenol-chloroform extraction, making the procedure simple and convenient. The purified plasmid DNA is suitable for downstream applications including restriction enzyme digestion, PCR, Sanger sequencing, and cell transfection. The processed product is intended for scientific research use only.
M1521549
| Component | 10T | 50T | Storage |
|---|
| M1521549A | RNase A | 40 mg | 200 mg | -20°C | | M1521549B | Magnetic Beads MXVB | 3.3 mL | 16.5 mL | 2–8°C | | M1521549C | Endotoxin Removal Buffer ERB | 154 mL | 770 mL | RT | | M1521549D | Endotoxin Removal Reagent ETR | 30 mL | 150 mL | RT | | M1521549E | Resuspension Buffer S1 | 112 mL | 560 mL | RT | | M1521549F | Lysis Buffer S2 | 112 mL | 560 mL | RT | | M1521549G | Precipitation Buffer N5 | 55 mL | 275 mL | RT | | M1521549H | Wash Buffer BW1 | 100 mL | 495 mL | RT | | M1521549I | Wash Buffer DW2 | 200 mL | 495 mL × 2 | RT | | M1521549J | Elution Buffer DE | 33 mL | 165 mL | RT |
|
Storage and Stability:
M1521549A: Store at -20°C, stable for 12 months.
M1521549B: Store at 2–8°C, stable for 12 months.
M1521549C, M1521549D, M1521549E, M1521549F, M1521549G, M1521549H, M1521549I, M1521549J: Store at room temperature (RT), stable for 12 months.
Compatible Instruments
Manual extraction.
Precautions
Read the entire manual carefully before use.
Several buffers in this kit contain irritating guanidine salts. Always wear gloves and follow standard safety precautions when handling. Avoid contact of buffers with skin, eyes, and mucous membranes. If contact occurs, wash immediately with plenty of water and seek medical attention.
If Lysis Buffer S2 develops a precipitate due to low ambient temperature, warm the buffer at 37°C in a water bath until the precipitate completely dissolves before use.
If the magnetic beads cannot be resuspended by vigorous shaking due to low temperature exposure, do not use the product.
Buffers in this kit contain guanidine salts. Do not treat with oxidizing disinfectants such as sodium hypochlorite, as this may release toxic gases. Dispose of waste according to medical waste regulations.
This kit is designed for use as an integrated system. Do not interchange or mix components from different lot numbers.
Protocol
1. Sample Requirements
Specimen Type: 50–200 mL bacterial culture.
Sample Storage and Transport: Samples can be tested immediately or stored at -70°C or lower for up to 6 months. Avoid repeated freeze-thaw cycles. Transport samples using cold chain logistics.
Freeze-Thaw Requirements: Snap-freeze and thaw rapidly. Avoid repeated freeze-thaw cycles.
2. Equipment and Materials Required but Not Provided
Constant-temperature water bath (for dissolving precipitates if necessary), dry block heater.
Magnetic separation rack for 1.5 mL centrifuge tubes, 15 mL centrifuge tubes, and 50 mL centrifuge tubes.
1.5/2.0 mL sterile, nuclease-free centrifuge tubes, adjustable pipettes and corresponding tips (20, 200, 1000 μL).
3. Reagent Preparation
Inspect all solutions for precipitates and ensure the magnetic beads can be fully resuspended.
For 10T Kit: Add 2 mL of Resuspension Buffer S1 to the vial containing RNase A dry powder. Mix by gentle inversion or vortexing to completely dissolve the RNase A. Transfer the entire dissolved RNase A solution back into the Resuspension Buffer S1 bottle. Mix well. Store at 2–8°C. Stable for 6 months after preparation.
For 50T Kit: Add 5 mL of Resuspension Buffer S1 to the vial containing RNase A dry powder. Mix by gentle inversion or vortexing to completely dissolve the RNase A. Transfer the entire dissolved RNase A solution back into the Resuspension Buffer S1 bottle. Mix well. Store at 2–8°C. Stable for 6 months after preparation.
4. Operating Procedure
Magnetic Separation Guidance (applicable from step 4.10 onwards): After placing the tube on the magnetic rack, gently rotate the tube left and right to allow the magnetic beads to collect against the tube wall nearest the magnet. Gently invert the rack several times to ensure beads on the cap also migrate to the wall. Let stand for several minutes until the solution becomes clear (standing time depends on the magnetic strength of the rack).
4.1 Add 50–200 mL of LB medium containing the appropriate antibiotic to a culture flask. Inoculate the medium with the bacterial strain containing the target vector. Incubate at 37°C with shaking for 12–16 hours.
Note: Use of LB medium is recommended. If using richer media such as TB or YT for bacterial culture, reduce the sample volume by half.
4.2 Harvest the bacterial cells by centrifugation at 10,000 × g for 1 minute using an appropriately sized centrifuge tube. For larger volumes (e.g., 200 mL culture), collect the cells by performing four sequential centrifugation runs using 50 mL centrifuge tubes. Decant the culture medium and gently tap the inverted tube on absorbent paper to remove residual liquid.
4.3 Add 10 mL of S1/RNase A mixture. Resuspend the bacterial pellet thoroughly by high-speed vortexing.
Note: Ensure the bacterial pellet is completely resuspended. Incompletely dispersed clumps will reduce lysis efficiency, resulting in lower yield and purity.
4.4 Add 10 mL of Lysis Buffer S2 to the resuspended cells. Mix by gentle inversion 2–3 times. Incubate at room temperature for 8–10 minutes, gently inverting the tube 20–30 times every 2 minutes during this period.
Note: Mix by gentle inversion only; vortexing will cause genomic DNA contamination. The solution should become viscous and clear, indicating complete lysis. If lysis with S2 is incomplete, consider reducing the starting amount of bacterial culture.
4.5 Immediately add 5 mL of Precipitation Buffer N5. Mix immediately by gently inverting 15–25 times to completely neutralize the solution.
Note: Invert immediately after adding N5 to prevent localized precipitation that hinders neutralization. When processing larger bacterial volumes, neutralization may be more difficult; shake slightly more vigorously several times until the solution is completely neutralized. After neutralization, the precipitate should appear white. If neutralization is incomplete, increase the number of inversions until fully neutralized.
4.6 Centrifuge at 10,000 × g for 5 minutes.
4.7 Choose one of the following protocols based on endotoxin level requirements:
Option ① (Rapid Extraction, suitable for standard transfection-grade plasmid; typical endotoxin level: ~200–500 EU/μg): No further treatment required. Proceed directly to step 4.8.
Option ② (Enhanced Endotoxin Removal, suitable for applications requiring lower endotoxin levels; typical endotoxin level: ~0.1–2.5 EU/μg): Transfer the supernatant to a new centrifuge tube. Add 1/10 volume of Endotoxin Removal Reagent ETR (e.g., add 60 μL ETR to 600 μL supernatant). Mix by inversion. Incubate at 4°C for 10 minutes, then transfer to 42°C and incubate for 10 minutes. Centrifuge at 10,000 × g for 3 minutes.
4.8 Transfer the entire supernatant to a new 50 mL centrifuge tube.
4.9 Add 0.7 volumes (relative to the supernatant volume) of Endotoxin Removal Buffer ERB and 300 μL of Magnetic Beads MXVB. Mix by vortexing for 5–7 minutes to allow DNA adsorption onto the beads.
4.10 Place the tube on the magnetic rack and allow separation until the magnetic collection is complete. Aspirate and discard the supernatant carefully, avoiding disturbance of the magnetic beads.
4.11 Add 9 mL of Wash Buffer BW1. Let stand to soak for 3–5 minutes. Remove the tube, mix by inversion for 30 seconds, then place back onto the 50 mL magnetic rack. Allow magnetic separation until the solution is clear. Aspirate and discard the supernatant, avoiding the beads.
Note: The soaking step is critical for consistent yield and should not be omitted.
4.12 Add 9 mL of Wash Buffer DW2. Vortex for 30 seconds to disperse the beads. Place on the 50 mL magnetic rack and allow separation until the solution is clear. Aspirate and discard the supernatant, avoiding the beads.
4.13 Add another 3 mL of Wash Buffer DW2. Vortex for 30 seconds to disperse the beads. Place on the 50 mL magnetic rack and allow separation until the solution is clear. Aspirate and discard the supernatant, avoiding the beads.
4.14 With the tube still on the magnetic rack, carefully aspirate any remaining liquid from the tube and cap. Air-dry the beads for 5–10 minutes.
Note: Residual ethanol can inhibit downstream enzymatic reactions; ensure complete evaporation of ethanol during drying. If liquid condenses and runs down to the bottom of the tube during drying, aspirate it carefully. Dry until the bead surface appears free of visible liquid. Avoid using an oven or other heat sources for drying, as excessive drying reduces DNA recovery during elution.
4.15 Add 1–3 mL of Elution Buffer DE. Use a pipette to rinse the tube wall with the elution buffer, ensuring any residual beads on the wall are washed down and collected at the bottom.
4.16 Transfer the elution buffer and magnetic beads to one or two 2.0 mL centrifuge tubes. Mix by vortexing for approximately 30 seconds.
4.17 Incubate the tube(s) at 60°C for 5 minutes, followed by high-speed vortexing for 30 seconds.
4.18 Briefly centrifuge to collect liquid droplets from the cap(s). Place the tube(s) on the magnetic rack for separation.
4.19 Transfer the DNA solution to a new centrifuge tube. If not used immediately, store at -15 to -25°C. For long-term storage, keep at -70°C or lower.
Limitations of the Method
For certain poorly collected or processed samples, trace impurities may be present in the extracted product that could inhibit PCR amplification.
The efficiency of nucleic acid extraction is highly dependent on the quality of sample collection, processing, transport, and storage. Errors in any of these steps may lead to inaccurate results.
ZJ26F0636040
危险品化学品经营许可证（带存储）