本试剂盒适用于从各种水体样本中提取高纯度的基因组DNA，如自来水，河水，养殖池塘水，污水等样本，该方法采用腐殖酸吸附剂和磁珠纯化技术。吸附剂可高效地吸附DNA溶液中的抑制因子。提取过程中无需使用有毒的酚氯仿抽提，也无需进行耗时的醇类沉淀。该方法纯化的DNA包括细菌、真菌等微生物的基因组DNA。得到的DNA可直接用于PCR、细菌DNA检测等实验。其处理后的产物用于科研检测使用。
| 产品号 | 组件名称 | 物理外观 | 48T | 储存温度 | | M1522592A | 裂解液AL | 液体 | 35 mL | RT. | | M1522592B | 磁珠MBVB | 液体 | 1.1 mL | 2-8℃. | | M1522592C | 研磨管 (ZB_M1) | — | 1 EA×48 | RT. | | M1522592D | RNase A | 粉末 | 6 mg | 2-8℃. | | M1522592E | 酶溶解液ESB | 液体 | 1 mL | RT. | | M1522592F | 沉淀液SPS | 液体 | 11 mL | RT. | | M1522592G | 裂解结合液BLS | 液体 | 30 mL | RT. | | M1522592H | 洗涤液AW1 | 液体 | 40 mL | RT. | | M1522592I | 洗涤液DW2 | 液体 | 80 mL | RT. | | M1522592J | 洗脱液DE | 液体 | 6 mL | RT. | | M1522592K | 蛋白酶K | 液体 | 1.1 mL | 2-8℃. |
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储存条件
M1522592B, M1522592D, M1522592K：2-8℃保存，12个月。
M1522592A, M1522592C, M1522592E, M1522592F, M1522592G, M1522592H, M1522592I, M1522592J：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AL、沉淀液SPS出现沉淀，可60℃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：各种水体样本，如自来水，河水，养殖池塘水，污水等样本。
- 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）、0.22 µm或0.45 µm滤膜
3. 试剂准备
4. 操作步骤
磁分离操作建议（步骤4.6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 用0.22 µm或0.45 µm滤膜过滤收集水体微生物，如果水样中不可溶解的颗粒较多，易造成堵塞，可在0.22 µm或0.45 µm 滤膜上放置一些更大孔径的滤膜，如在滤膜上放置一层1 µm滤膜和一层3~8 µm滤膜，减少堵塞现象。
4.2 将0.22 µm或0.45 µm滤膜取出，剪成1~2 mm的碎片至研磨管 (ZB_M1) 中，加入600 µL裂解液AL和20 µL蛋白酶K，涡旋仪最大转速涡旋或转移至震荡破碎仪高速研磨样本10分钟。（振荡破碎仪中选择合适的程序进行裂解，例如MP公司FastPrep-24，4℃ 6.5m/sec，on 90 sec，off 3 min，2 cycles。如使用其他品牌振荡破碎仪，请选择仪器推荐程序。）
4.3 加入10 µL RNase A涡旋混匀，70℃温浴20~30分钟，涡旋混匀30秒。
4.4 视溶液腐殖酸含量高低进行以下处理（二选一）
- 对于腐殖酸含量较低的样本：13,000×g离心5分钟，按步骤4.5取上清进行操作。
- 对于腐殖酸含量较高的样本：加入200 µL沉淀液SPS，涡旋混匀30秒，4℃冰箱或冰上放置5~10分钟，13,000 ×g离心5分钟。（注意：沉淀液去腐殖酸的同时会降低DNA产量，对于腐殖酸含量低的样本，建议省略此步骤。）
4.5 转移500 µL上清至新的离心管中，分别加入20 µL磁珠MBVB，500 μL裂解结合液BLS，振荡或涡旋5~7分钟使磁珠吸附DNA。
4.6 转移至磁力架上进行磁分离至溶液澄清，吸弃溶液。
4.7 加入700 µL洗涤液AW1，涡旋30秒后转至磁力架上进行磁分离至溶液澄清，吸弃溶液。
4.8 加入700 µL洗涤液DW2，涡旋30秒后转至磁力架上进行磁分离至溶液澄清，吸弃溶液。
4.9 再次加入700 µL洗涤液DW2，涡旋30秒后转至磁力架上进行磁分离至溶液澄清，吸弃溶液。
4.10 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。
4.11 加入50~100 µL洗脱液DE，高速涡旋2~3分钟打散磁珠。
4.12 60℃温浴10分钟，温浴期间取出涡旋打散磁珠2~3次。
4.13 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至溶液澄清。
4.14 把基因组DNA溶液转移至新的离心管中。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 suitable for extracting high‑purity genomic DNA from various water samples, such as tap water, river water, aquaculture pond water, and wastewater. The method employs humic acid adsorbent and magnetic bead purification technology. The adsorbent efficiently removes inhibitors from the DNA solution. The extraction process avoids toxic phenol‑chloroform extraction and time‑consuming alcohol precipitation. The nucleic acids purified by this method include genomic DNA from bacteria, fungi, and other microorganisms. The obtained DNA can be used directly in PCR, bacterial DNA detection, and other experiments. The purified DNA is intended for research use only.
| Product Code | Component | Appearance | 48T | Storage |
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| M1522592A | Lysis Buffer AL | Liquid | 35 mL | RT | | M1522592B | Magnetic Beads MBVB | Liquid | 1.1 mL | 2-8 °C | | M1522592C | Grinding Tube (ZB_M1) | – | 1 EA×48 | RT
| | M1522592D | RNase A | Powder | 6 mg | 2-8 °C | | M1522592E | Enzyme Dissolution Buffer ESB | Liquid | 1 mL | RT
| | M1522592F | Precipitation Solution SPS | Liquid | 11 mL | RT
| | M1522592G | Lysis/Binding Buffer BLS | Liquid | 30 mL | RT
| | M1522592H | Wash Buffer AW1 | Liquid | 40 mL | RT
| | M1522592I | Wash Buffer DW2 | Liquid | 80 mL | RT
| | M1522592J | Elution Buffer DE | Liquid | 6 mL | RT
| | M1522592K | Proteinase K | Liquid | 1.1 mL | 2-8 °C |
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Storage Conditions
M1522592B, M1522592D, M1522592K: Store at 2–8 °C for 12 months.
M1522592A, M1522592C, M1522592E, M1522592F, M1522592G, M1522592H, M1522592I, M1522592J: Store at room temperature for 12 months.
Compatible Instrumentation
Manual extraction.
Precautions
Read the entire manual carefully before use.
Several buffers contain irritating guanidine salts. Always wear gloves and follow standard safety precautions. Avoid contact with skin, eyes, and mucous membranes. If contact occurs, rinse immediately with plenty of water and seek medical attention.
If precipitates appear due to low temperature, warm the solution at 30 °C until completely dissolved before use.
If precipitates appear in Lysis Buffer AL or Precipitation Solution SPS due to low temperature, warm at 60 °C until completely dissolved before use.
If magnetic beads cannot be resuspended by vigorous shaking due to low temperature, do not use.
Several buffers contain guanidine salts. Do not treat with oxidizing disinfectants (e.g., sodium hypochlorite) as this may release toxic gas. Dispose as medical waste.
This kit is intended for use as an integrated system. Do not interchange or mix components from different lot numbers.
Protocol
1. Sample Requirements
Applicable sample types: Various water samples, such as tap water, river water, aquaculture pond water, and wastewater.
Sample preservation and transport: Samples can be processed immediately or stored at –70 °C or lower for up to 6 months. Avoid repeated freeze‑thaw cycles. Transport samples under cold chain conditions.
Freeze‑thaw requirements: Quick freeze and quick thaw; avoid repeated freeze‑thaw cycles.
2. Materials to Be Prepared by User
Water bath (for dissolving precipitates), dry thermostat bath
Magnetic stand (for 1.5/2.0 mL tubes)
1.5/2.0 mL sterile, nuclease‑free centrifuge tubes
Various pipettes and tips (20 µL, 200 µL, 1000 µL)
0.22 µm or 0.45 µm filter membrane
3. Reagent Preparation
Check solutions for precipitates and ensure magnetic beads can be resuspended.
Dissolve RNase A (10 mg/mL): Add 0.6 mL of Enzyme Dissolution Buffer ESB to the RNase A powder to obtain a final concentration of 10 mg/mL. Mix by inversion or gentle vortexing until fully dissolved. The dissolved RNase A should be stored at –20 °C to 8 °C.
4. Procedure
Recommended magnetic separation procedure (starting from step 4.6): Place the tube on the magnetic stand, gently rotate left and right until the beads aggregate on the tube wall closest to the magnet. Gently invert the magnetic stand several times to collect beads from the cap onto the wall. Let stand for several minutes until the solution is clear (stand time depends on the magnet strength).
4.1 Collect microorganisms from the water sample by filtration using a 0.22 µm or 0.45 µm filter membrane. If the water contains a high amount of insoluble particles that may cause clogging, place a larger‑pore membrane (e.g., 1 µm and 3–8 µm) on top of the 0.22 µm or 0.45 µm membrane to reduce clogging.
4.2 Remove the 0.22 µm or 0.45 µm filter membrane, cut it into 1–2 mm pieces, and transfer them into a Grinding Tube (ZB_M1). Add 600 μL of Lysis Buffer AL and 20 μL of Proteinase K. Vortex at maximum speed or transfer to a bead beater and grind at high speed for 10 minutes. (Select an appropriate program for lysis in the bead beater; for example, MP Biomedicals FastPrep‑24: 4 °C, 6.5 m/sec, on 90 sec, off 3 min, 2 cycles. For other brands, follow the instrument‑recommended program.)
4.3 Add 10 μL of RNase A (10 mg/mL), vortex to mix, then incubate at 70 °C for 20–30 minutes. Vortex for 30 seconds.
4.4 Based on the humic acid content of the sample, choose one of the following two options:
For samples with low humic acid content: Centrifuge at 13,000×g for 5 minutes and proceed to step 4.5 using the supernatant.
For samples with high humic acid content: Add 200 μL of Precipitation Solution SPS, vortex for 30 seconds, then place at 4 °C (refrigerator or on ice) for 5–10 minutes. Centrifuge at 13,000×g for 5 minutes. (Note: The precipitation solution removes humic acids but may reduce DNA yield; for low‑humic‑acid samples, it is recommended to skip this step.)
4.5 Transfer 500 μL of the supernatant to a new centrifuge tube. Add 20 μL of Magnetic Beads MBVB and 500 μL of Lysis/Binding Buffer BLS. Shake or vortex for 5–7 minutes to allow the beads to bind DNA.
4.6 Place the tube on a magnetic stand for magnetic separation until the solution is clear. Aspirate and discard the supernatant.
4.7 Add 700 μL of Wash Buffer AW1. Vortex for 30 seconds, then return to the magnetic stand and separate until clear. Aspirate and discard the supernatant.
4.8 Add 700 μL of Wash Buffer DW2. Vortex for 30 seconds, then separate magnetically until clear. Aspirate and discard the supernatant.
4.9 Add another 700 μL of Wash Buffer DW2. Vortex for 30 seconds, then separate magnetically until clear. Aspirate and discard the supernatant.
4.10 Briefly centrifuge in a microcentrifuge. Place the tube on the magnetic stand and allow the solution to become clear. Aspirate and discard all remaining liquid thoroughly. Open the cap and air‑dry for 3–5 minutes.
Note: Residual ethanol will inhibit downstream enzymatic reactions. Ensure that ethanol is completely evaporated during drying. Do not over‑dry, as this may affect elution efficiency.
4.11 Add 50–100 μL of Elution Buffer DE. Vortex vigorously for 2–3 minutes to fully resuspend the beads.
4.12 Incubate at 60 °C for 10 minutes. During incubation, remove the tube and vortex to resuspend the beads 2–3 times.
4.13 Briefly centrifuge to collect droplets from the cap. Return the tube to the magnetic stand for magnetic separation until the solution is clear.
4.14 Transfer the genomic 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 samples, the extracted product may contain trace impurities that inhibit PCR amplification.
The efficiency of nucleic acid extraction is closely related to sample collection, processing, transport, and storage. Any error in these steps may lead to inaccurate results.
ZJ26F0636847
危险品化学品经营许可证（带存储）