本试剂盒提供了一种从粪便样本中提取总DNA的方法，可提取样本中细菌、寄生虫以及病毒的总DNA，同时适用于含有高浓度PCR反应抑制剂的样本DNA提取。本试剂盒采用独特的缓冲系统，使裂解液中的DNA高效结合到吸附柱上；PCR和酶反应的抑制剂以及残留的杂质可通过洗涤步骤有效去除，最后使用低盐缓冲液（Buffer EB）或灭菌水洗脱，即可获得高纯度DNA。纯化得到的DNA可直接用于二代测序（16S扩增子和宏基因组）、文库构建、PCR、qPCR、Southern Blot和酶切分子标记等下游实验。
产品号
| 组件
| 物理外观
| 50T
| 储存温度
|
F1522829A
| Buffer MS1
| 液体
| 33 mL | RT.
|
F1522829B
| Buffer MS2
| 液体
| 10 mL | RT.
|
F1522829C
| Buffer NDS3
| 液体
| 10 mL | RT.
|
F1522829D
| Buffer SGB
| 液体
| 44 mL | RT.
|
F1522829E
| Buffer PW1
| 液体
| 17 mL | RT.
|
F1522829F
| Buffer PW2
| 液体
| 15 mL | RT.
|
F1522829G
| Buffer EB
| 液体
| 15 mL | RT.
|
F1522829H
| 蛋白酶K
| 液体
| 1.2 mL | RT.
|
F1522829I
| RNase A (10 mg/mL)
| 液体
| 550 μL | RT.
|
F1522829J
| 吸附柱PBF (含2mL收集管)
| —
| 1 EA×50
| RT.
|
F1522829K
| 匀浆管A
| —
| 1 EA×50
| RT. |
试剂盒置于室温干燥条件下可保存15个月。
注意事项
1. 新采取的样本会得到更高的得率，不同样本在采样前应先查阅相应的最佳保存条件。
2. 在需要吸取上清液的步骤中应避免吸到沉淀，否则会影响产物纯度。
3. 过量的DNA可能抑制下游PCR反应，遇到这种情况建议将DNA模板进行稀释后使用。
4. 使用前检查Buffer MS2是否有沉淀，若有沉淀，请在37℃加热至完全溶解后使用。
操作步骤
使用前请先在Buffer PW1中加入22 mL无水乙醇，Buffer PW3中加入60 mL的无水乙醇。
1. 在匀浆管A中加入100-300 mg粪便样本；若为液态粪便样本，则转移200 µL至匀浆管中。
2. 向匀浆管中加入600 µL Buffer MS1、160 µL Buffer MS2和20 µL 蛋白酶K；若为干粪便样本，可按1.5-2倍的比例酌情增加Buffer MS1和Buffer MS2的用量。
3. 将匀浆管放于70℃、1500 rpm的恒温混匀仪上振荡混匀15 min。若使用振荡研磨装置，则将离心管固定在装有2 mL适配器的振荡研磨装置中，并根据设备使用说明优化研磨条件进行处理（具体方法参见附录）。
4. 12,000 rpm (13,680 × g) 离心2 min，小心转移上清液至新的2 mL离心管中（避免吸到沉淀）；加入10 µL RNase A，短暂涡旋振荡混合，室温放置5 min，去除RNA杂质。
5. 向上述离心管中加入160 µL Buffer NDS3，充分混匀，涡旋振荡5 sec，置于4℃环境中放置5 min，进一步去除PCR抑制剂。
6. 12,000 rpm (13,680 × g) 离心2 min，使杂质充分沉淀。
7. 转移550 μL上清液到新的2 mL离心管中（若上清液不足550 μL，转移全部上清即可）；加入800 μL Buffer SGB，涡旋振荡混匀5 sec后，将离心管放于25℃、1500 rpm的恒温混匀仪上振荡混匀5 min，或连续颠倒混匀5 min，促进DNA与Buffer SGB结合。【注意】转移上清时不要移走沉淀，否则可能降低DNA纯度。
8. 将步骤7得到的溶液和沉淀一起转入吸附柱PBF（将吸附柱放入配套的2 mL收集管中），12,000 rpm (13,680 × g) 离心30 sec，弃去收集管中的废液，将吸附柱重新放回收集管中。每次加入量不超过700 μL，若体积超出可分多次转入，直至所有液体转移完成。
9. 向吸附柱中加入700 μL Buffer PW1（使用前已加入无水乙醇），12,000 rpm (13,680×g) 离心30 sec，弃去废液，将吸附柱放回收集管中。
10. 向吸附柱中加入700 μL Buffer PW2（使用前已加入无水乙醇），12,000 rpm (13,680×g) 离心30 sec，弃去废液，将吸附柱放回收集管中。
11. 重复步骤10（第二次洗涤）的操作，进一步去除吸附柱上残留的抑制剂、盐分等杂质，提升DNA纯度。
12. 将吸附柱连同收集管一起，12,000 rpm (13,680 × g) 离心2 min，彻底去除吸附柱中残留的洗涤液和杂质（避免残留乙醇影响后续DNA洗脱和实验）。
13. 将吸附柱从收集管中取出，转入一个新的1.5 mL离心管中，开盖放置2 min，彻底挥发吸附柱中残留的乙醇。向吸附膜中间部位缓慢悬空滴加50 μL Buffer EB，室温放置1 min；12,000 rpm (13,680×g) 离心30 sec，收集离心管中的DNA溶液，可立即用于后续实验，或置于-20℃长期保存。
注意：
(1) 如果下游实验对pH值或EDTA敏感，可以用灭菌水洗脱。洗脱液的pH值对洗脱效率有很大影响，若用水做洗脱液应保证其pH值在7.0-8.5（可以用NaOH将水的pH值调到此范围），pH值低于7.0时洗脱效率不高。
(2) 如果要提高DNA的终浓度，可以将步骤13所得的DNA洗脱液重新加至吸附膜上，室温放置2 min，12,000 rpm (13,680×g) 离心30sec。
(3) 如需长期保存，建议使用Buffer EB洗脱并于-20℃长期保存，可有效防止DNA降解。
附录：样本研磨方法
可选择以下方法之一研磨样本，确保样本充分破碎，提升DNA提取效率：
1. 在涡旋振荡仪上以最大速度手动或自动涡旋振荡10 min。
2. 在搭配有1.5-2 mL水平离心管支架的涡旋振荡仪上，以最大速度振荡10 min（研磨管保持水平放置）；若样本数量超过12个，延长振荡时间5-10 min（例如使用Scientific Industries或Mobio的Vortex-Genie2涡旋振荡仪）。
3. 使用Qiagen的TissueLyser II时，以25 Hz研磨10 min。
4. 使用Qiagen的PowerLyzer 24 Homogenizer时，以2000 rpm的速度均质化30 sec，暂停30 sec，然后以2000 rpm的速度再次均质化30 sec。
5. 使用MP Biomedicals的FastPrep-24时，推荐速度为6.0，时间为40 sec。
DNA浓度及纯度检测
提取的基因组DNA片段大小，与样品保存时间、操作过程中的剪切力等因素有关。纯化得到的DNA片段可用琼脂糖凝胶电泳和紫外分光光度计两种方法，检测其浓度与纯度。
1. 浓度检测
通过紫外分光光度计测量样品在260 nm处的吸光度（A260），确定DNA浓度。若需稀释样品，建议使用Buffer EB进行稀释，用水稀释样本可能导致数值不准确。260 nm处准确的吸光度应在0.1-1.0范围内，以保证检测结果的准确性。换算标准：260 nm处1个单位的吸光度，相当于50µg/mL双链DNA，或40µg/mL单链DNA。
2. 纯度检测
通过检测A260/A280的比值判断DNA纯度，合格的基因组DNA，其A260/A280比值应在1.7-1.9之间。如果洗脱时未使用Buffer EB，而是使用ddH₂O，比值会偏低，因为pH值和离子存在会影响光吸收值，但并不表示DNA纯度低。
This kit provides a method for extracting total DNA from fecal samples, enabling the extraction of total DNA from bacteria, parasites, and viruses present in the sample. It is also suitable for extracting DNA from samples containing high concentrations of PCR reaction inhibitors. The kit uses a unique buffer system that allows efficient binding of DNA from the lysate to the spin column. Inhibitors of PCR and enzymatic reactions, as well as residual impurities, are effectively removed through washing steps. Finally, high‑purity DNA is eluted with a low‑salt buffer (Buffer EB) or sterile water. The purified DNA can be directly used in downstream applications such as next‑generation sequencing (16S amplicon and metagenomics), library construction, PCR, qPCR, Southern blot, and restriction enzyme‑based molecular markers.
| Product No. | Component | Appearance | 50T | Storage |
|---|
| F1522829A | Buffer MS1 | Liquid | 33 mL | RT | | F1522829B | Buffer MS2 | Liquid | 10 mL | RT | | F1522829C | Buffer NDS3 | Liquid | 10 mL | RT | | F1522829D | Buffer SGB | Liquid | 44 mL | RT | | F1522829E | Buffer PW1 | Liquid | 17 mL | RT | | F1522829F | Buffer PW2 | Liquid | 15 mL | RT | | F1522829G | Buffer EB | Liquid | 15 mL | RT | | F1522829H | Proteinase K | Liquid | 1.2 mL | RT | | F1522829I | RNase A (10 mg/mL) | Liquid | 550 μL | RT | | F1522829J | PBF Spin Column (with 2 mL collection tube) | — | 1 EA × 50 | RT | | F1522829K | Homogenization Tube A | — | 1 EA × 50 | RT |
|
The kit can be stored under dry conditions at room temperature for 15 months.
Precautions
Freshly collected samples yield higher recovery. Before sampling, consult the appropriate optimal storage conditions for different sample types.
When pipetting supernatants, avoid sucking up any precipitate, as this may affect product purity.
Excess DNA may inhibit downstream PCR reactions. If this occurs, it is recommended to dilute the DNA template before use.
Before use, check Buffer MS2 for precipitation. If precipitation is present, heat at 37°C until completely dissolved before use.
Procedure
Before use, add 22 mL of absolute ethanol to Buffer PW1 and 60 mL of absolute ethanol to Buffer PW3.
1. Add 100–300 mg of fecal sample to Homogenization Tube A. For liquid fecal samples, transfer 200 µL to the tube.
2. Add 600 µL of Buffer MS1, 160 µL of Buffer MS2, and 20 µL of Proteinase K to the tube. For dry fecal samples, the amounts of Buffer MS1 and Buffer MS2 can be increased proportionally by a factor of 1.5–2.
3. Place the homogenization tube on a thermomixer at 70°C and 1500 rpm, and shake for 15 min. If using a bead‑beating device, secure the tube in a 2‑mL adapter and process according to the device instructions (see Appendix for details).
4. Centrifuge at 12,000 rpm (13,680 × g) for 2 min. Carefully transfer the supernatant to a new 2 mL centrifuge tube (avoiding any precipitate). Add 10 µL of RNase A, vortex briefly to mix, and let stand at room temperature for 5 min to remove RNA impurities.
5. Add 160 µL of Buffer NDS3 to the tube, mix thoroughly, vortex for 5 sec, and place at 4°C for 5 min to further remove PCR inhibitors.
6. Centrifuge at 12,000 rpm (13,680 × g) for 2 min to fully pellet impurities.
7. Transfer 550 μL of the supernatant to a new 2 mL centrifuge tube (if the supernatant volume is less than 550 μL, transfer all of it). Add 800 μL of Buffer SGB, vortex for 5 sec, then place the tube on a thermomixer at 25°C and 1500 rpm and shake for 5 min, or invert continuously for 5 min to promote DNA binding to Buffer SGB.
【Note】 Do not transfer any precipitate when removing the supernatant, as this may reduce DNA purity.
8. Transfer the solution and precipitate from step 7 into a PBF spin column (placed in the provided 2 mL collection tube). Centrifuge at 12,000 rpm (13,680 × g) for 30 sec. Discard the flow‑through and return the spin column to the collection tube. Add no more than 700 μL per transfer; if the volume exceeds, transfer in multiple portions until all liquid has been loaded.
9. Add 700 μL of Buffer PW1 (with ethanol pre‑added) to the spin column. Centrifuge at 12,000 rpm (13,680 × g) for 30 sec. Discard the flow‑through and return the spin column to the collection tube.
10. Add 700 μL of Buffer PW2 (with ethanol pre‑added) to the spin column. Centrifuge at 12,000 rpm (13,680 × g) for 30 sec. Discard the flow‑through and return the spin column to the collection tube.
11. Repeat step 10 (second wash) to further remove residual inhibitors, salts, and other impurities on the spin column, thereby increasing DNA purity.
12. Centrifuge the spin column together with the collection tube at 12,000 rpm (13,680 × g) for 2 min to completely remove residual wash buffer and impurities (this avoids residual ethanol affecting subsequent DNA elution and experiments).
13. DNA elution
Transfer the spin column to a new 1.5 mL centrifuge tube. Open the cap and let stand for 2 min to allow residual ethanol to evaporate completely. Slowly add 50 μL of Buffer EB dropwise to the center of the membrane without touching it. Let stand at room temperature for 1 min. Centrifuge at 12,000 rpm (13,680 × g) for 30 sec. Collect the DNA solution in the centrifuge tube. The DNA can be used immediately for downstream experiments or stored at –20°C for long‑term preservation.
Notes:
(1) If downstream applications are sensitive to pH or EDTA, sterile water can be used for elution. The pH of the elution buffer significantly affects elution efficiency. If water is used, ensure its pH is between 7.0 and 8.5 (adjust with NaOH). pH values below 7.0 will reduce elution efficiency.
(2) To increase the final DNA concentration, reload the collected DNA eluate from step 13 onto the same membrane, let stand at room temperature for 2 min, and centrifuge at 12,000 rpm (13,680 × g) for 30 sec.
(3) For long‑term storage, it is recommended to elute with Buffer EB and store at –20°C to effectively prevent DNA degradation.
Appendix: Sample Grinding Methods
Choose one of the following methods to grind the sample, ensuring thorough disruption to improve DNA extraction efficiency:
Vortex manually or automatically at maximum speed on a vortex mixer for 10 min.
Using a vortex mixer equipped with a 1.5–2 mL horizontal centrifuge tube holder, vortex at maximum speed for 10 min (keep the tube horizontal). If more than 12 samples are processed, extend the vortexing time by 5–10 min (e.g., using a Vortex‑Genie 2 from Scientific Industries or Mobio).
When using a Qiagen TissueLyser II, grind at 25 Hz for 10 min.
When using a Qiagen PowerLyzer 24 Homogenizer, homogenize at 2000 rpm for 30 sec, pause for 30 sec, then homogenize again at 2000 rpm for 30 sec.
When using an MP Biomedicals FastPrep‑24, recommended speed is 6.0 for 40 sec.
DNA Concentration and Purity Assessment
The fragment size of the extracted genomic DNA is influenced by factors such as sample storage time and shear forces during handling. The purified DNA can be assessed for concentration and purity by agarose gel electrophoresis and UV spectrophotometry.
1. Concentration determination
Measure the absorbance of the sample at 260 nm (A<sub>260</sub>) using a UV spectrophotometer to determine DNA concentration. If dilution is required, Buffer EB is recommended. Dilution with water may lead to inaccurate readings. The absorbance at 260 nm should be kept within 0.1–1.0 to ensure accuracy. Conversion: 1 unit of absorbance at 260 nm corresponds to 50 µg/mL of double‑stranded DNA or 40 µg/mL of single‑stranded DNA.
2. Purity assessment
Evaluate DNA purity by the A<sub>260</sub>/A<sub>280</sub> ratio. For high‑quality genomic DNA, the A<sub>260</sub>/A<sub>280</sub> ratio should be between 1.7 and 1.9. If ddH₂O instead of Buffer EB is used for elution, the ratio may be lower due to the influence of pH and ions on light absorption; this does not necessarily indicate low DNA purity.
ZJ26F0636846
危险品化学品经营许可证（带存储）