本试剂盒适用于从1~5 mL细菌培养液中提取高达20 µg的质粒DNA，该方法采用超顺磁性的磁性粒子纯化技术，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转化、酶切、PCR、一代测序和文库筛选等相关实验。其处理后的产物用于科研检测使用。
储存条件
M1521621A：-20℃保存，12个月。
M1521621B：2-8℃保存，12个月。
M1521621C, M1521621D, M1521621E, M1521621F, M1521621G, M1521621H, M1521621I：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，溶液出现沉淀，需要30℃水浴至沉淀完全溶解后方可使用。
4. 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。
5. 如因气温较低，用力振荡后磁珠无法重悬，请勿使用。
6. 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。
7. 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：1~5 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
- 恒温水浴锅（当溶液出现沉淀时使用）、干式恒温仪
- 磁力架（1.5/2.0 mL）、1.5/2.0 mL 无菌无核酸酶离心管、各种规格移液器及Tips（20、200、1000 μL）
3. 试剂准备
4. 操作步骤
磁分离操作建议（步骤4.9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10~20 mL培养管中加入1~5 mL含抗生素的LB培养液，将含目的载体的菌种接种至培养管中，37℃摇床培养12~16小时。
注意：不建议使用TB、YT等丰富培养基来培养细菌。使用丰富培养基，会导致RNA去除不干净。
4.2 10,000×g离心1分钟，收集1~5 mL菌体。
4.3 倒弃培养基，在吸水纸上轻轻拍打吸尽残液。加入250 μL 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250 μL裂解液S2，颠倒混匀8~10次，室温放置1~2分钟。
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250 μL沉淀液N3，立即颠倒混匀8~10次让溶液彻底中和。
注意：加入沉淀液N3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，中和时需要稍用力颠倒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 转移600 μL上清液至新的1.5 mL离心管中。
4.8 加入400 μL结合液Bind，20 μL磁珠MXVB，振荡或涡旋5~7分钟使磁珠吸附DNA。
4.9 转移至磁力架上磁分离至溶液澄清，吸弃溶液。
4.10 加入700 μL洗涤液BW1，浸泡30秒再涡旋打散磁珠，转至磁力架上进行磁分离至溶液澄清，吸弃溶液。
4.11 加入700 μL洗涤液DW2，涡旋30秒打散磁珠后转至磁力架上进行磁分离至溶液澄清，吸弃溶液。
4.12 再次加入700 μL洗涤液DW2，涡旋30秒打散磁珠后转至磁力架上进行磁分离至溶液澄清，吸弃溶液。
4.13 掌上离心机瞬时离心后，转至磁力架上吸附至溶液澄清，充分吸弃所有溶液，打开管盖，空气干燥3~5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。切勿干燥过久，以免影响后续洗脱效果。
4.14 加入50~80 μL洗脱液DE，高速涡旋2~3分钟打散磁珠。
4.15 60℃温浴3~5分钟，然后高速涡旋30秒。
4.16 瞬时离心收集管盖液滴至管中，转移至磁力架上吸附1~3分钟。
4.17 把质粒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 up to 20 µg of plasmid DNA from 1–5 mL of bacterial culture. The method employs superparamagnetic particle purification technology. The extraction process eliminates the need for toxic phenol-chloroform extraction. The procedure is simple and convenient, and the purified plasmid DNA can be used directly in downstream applications such as transformation, restriction enzyme digestion, PCR, first-generation sequencing, and library screening. The processed products are intended for scientific research and testing purposes only.
Storage Conditions
M1521621A: Store at -20°C, stable for 12 months.
M1521621B: Store at 2–8°C, stable for 12 months.
M1521621C, M1521621D, M1521621E, M1521621F, M1521621G, M1521621H, M1521621I: Store at room temperature, stable for 12 months.
Compatible Instruments
Manual extraction.
Precautions
Read this manual carefully before use.
Several buffers in this kit contain irritating guanidinium salts. Always wear gloves and handle according to standard safety precautions. Avoid contact of buffers with skin, eyes, and mucous membranes. In case of contact, flush immediately with plenty of water and seek medical attention.
If precipitation occurs in the solution due to low ambient temperatures, incubate at 30°C in a water bath until the precipitate is completely dissolved before use.
If precipitation occurs in Lysis Buffer S2 due to low ambient temperatures, incubate at 37°C in a water bath until the precipitate is completely dissolved before use.
If the magnetic beads cannot be resuspended after vigorous shaking due to low ambient temperatures, do not use the beads.
Several buffers in this kit contain guanidinium 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 components are designed to be used together as a system. Components from different lot numbers should not be interchanged or mixed.
Instructions for Use
1. Sample Requirements
Applicable sample type: 1–5 mL bacterial culture.
Sample storage and transport: Samples may be processed 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: Rapid freezing and rapid thawing are recommended; avoid repeated freeze-thaw cycles.
2. Equipment and Materials to be Supplied by User
Constant temperature water bath (for use when precipitation occurs in solutions)
Dry block heater
Magnetic separation rack (for 1.5/2.0 mL tubes)
1.5/2.0 mL sterile, nuclease-free centrifuge tubes
Pipettes and tips of various volumes (20, 200, 1000 μL)
3. Reagent Preparation
Check solutions for any precipitation and confirm that the magnetic beads can be resuspended.
Add 0.5–1 mL of Resuspension Buffer S1 to the RNase A dry powder vial. Mix by inversion or gentle vortexing to fully dissolve the RNase A. Transfer the entire RNase A solution back into the Resuspension Buffer S1 bottle and mix well. Store at 2–8°C; stable for 6 months after reconstitution.
4. Operating Procedure
Recommendation for magnetic separation (starting from step 4.9): After placing the centrifuge tube on the magnetic rack, gently rotate it left and right. Once the magnetic beads have aggregated against the tube wall closest to the magnet, gently invert the entire rack several times to collect any beads from the cap onto the tube wall. Allow to stand for several minutes until the solution becomes clear (standing time depends on the strength of the magnetic rack).
4.1 Add 1–5 mL of LB medium containing the appropriate antibiotic to a 10–20 mL culture tube. Inoculate the medium with the bacterial strain harboring the target vector and incubate at 37°C with shaking for 12–16 hours.
Note: The use of rich media such as TB or YT for bacterial culture is not recommended. Using rich media may result in incomplete removal of RNA.
4.2 Centrifuge at 10,000 × g for 1 minute to pellet 1–5 mL of bacterial cells.
4.3 Discard the supernatant completely. Gently tap the inverted tube on absorbent paper to remove residual liquid. Add 250 μL of the prepared S1/RNase A mixture and resuspend the bacterial pellet thoroughly by high-speed vortexing.
Note: It is essential to completely resuspend the bacterial pellet. Incompletely dispersed clumps will affect lysis efficiency, leading to reduced yield and purity.
4.4 Add 250 μL of Lysis Buffer S2 to the resuspended cells. Mix by gently inverting the tube 8–10 times. Incubate at room temperature for 1–2 minutes.
Note: Mix gently by inversion. Vortexing will cause genomic DNA contamination. The solution should become viscous and translucent, indicating complete bacterial lysis. If lysis with S2 is incomplete, it is recommended to reduce the initial amount of bacterial culture used.
4.5 Immediately add 250 μL of Precipitation Buffer N3. Mix by gently inverting the tube 8–10 times to thoroughly neutralize the solution.
Note: Mix immediately upon adding N3 to prevent localized precipitation that may hinder neutralization. When larger volumes of bacterial culture are used, neutralization may be more difficult; invert with slightly more force several times until the solution is completely neutralized. The precipitate after neutralization should appear white. If neutralization is incomplete, increase the number of inversion cycles until fully neutralized.
4.6 Centrifuge at 10,000 × g for 5 minutes.
4.7 Transfer 600 μL of the supernatant to a new 1.5 mL centrifuge tube.
4.8 Add 400 μL of Binding Buffer Bind and 20 μL of Magnetic Beads MXVB. Shake or vortex for 5–7 minutes to allow DNA binding to the beads.
4.9 Place the tube on the magnetic rack for magnetic separation until the solution is clear, then aspirate and discard the supernatant.
4.10 Add 700 μL of Wash Buffer BW1. Allow to soak for 30 seconds, then vortex to disperse the beads. Place on the magnetic rack for separation until the solution is clear. Aspirate and discard the supernatant.
4.11 Add 700 μL of Wash Buffer DW2. Vortex for 30 seconds to disperse the beads, then place on the magnetic rack for separation until the solution is clear. Aspirate and discard the supernatant.
4.12 Add another 700 μL of Wash Buffer DW2. Vortex for 30 seconds to disperse the beads, then place on the magnetic rack for separation until the solution is clear. Aspirate and discard the supernatant.
4.13 Perform a quick spin using a mini centrifuge, then place the tube back on the magnetic rack and allow beads to collect until the solution is clear. Carefully aspirate and discard all residual liquid. Open the tube cap and air-dry the beads for 3–5 minutes.
Note: Residual ethanol will inhibit downstream enzymatic reactions. Ensure ethanol has completely evaporated during drying. Do not over-dry the beads, as this may affect subsequent elution efficiency.
4.14 Add 50–80 μL of Elution Buffer DE and vortex at high speed for 2–3 minutes to fully disperse the beads.
4.15 Incubate at 60°C for 3–5 minutes, then vortex at high speed for 30 seconds.
4.16 Briefly centrifuge to collect any droplets from the cap into the tube, then place on the magnetic rack and allow to stand for 1–3 minutes.
4.17 Transfer the plasmid DNA solution to a new centrifuge tube for subsequent use. If not used immediately, store at -15 to -25°C. For long-term storage, place at -70°C or lower.
Limitations of the Method
For certain poorly collected samples, trace impurities in the extracted product may inhibit PCR amplification.
The efficiency of nucleic acid extraction is closely related to the quality of sample collection, handling, transport, and storage. Any error in these processes may lead to inaccurate results.
ZJ26F0636132
危险品化学品经营许可证（带存储）