本试剂盒采用独特的脱腐缓冲液系统，可最大限度去除土壤样本中的腐殖酸等干扰杂质；同时配套匀浆管，其内含的研磨珠可有效破碎土壤样本中的各种复杂成分，保证从土壤中提取的基因组DNA的完整性。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、酶切和分子标记等下游实验。
产品号
| 组件
| 物理外观
| 50T
| 储存温度
|
S1522826A
| Buffer MS1
| 液体
| 40 mL | RT.
|
S1522826B
| Buffer MS2
| 液体
| 4 mL | RT.
|
S1522826C
| Buffer MS3
| 液体
| 14 mL | RT.
|
S1522826D
| Buffer MS4
| 液体
| 11 mL | RT.
|
S1522826E
| Buffer MS5
| 液体
| 66 mL | RT.
|
S1522826F
| Buffer PWS1
| 液体
| 17 mL | RT.
|
S1522826G
| Buffer EB
| 液体
| 15 mL | RT.
|
S1522826H
| 匀浆管A
| —
| 1 EA×50
| RT.
|
S1522826I
| 吸附柱PBF (含2mL收集管)
| —
| 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，不同土壤样本在采样前应先查阅相应的最佳保存条件，避免样本变质影响提取效果。
2. 在需要吸取上清液的步骤中应避免吸到沉淀，否则会引入杂质，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。
5. 实验需自备无水乙醇（用于Buffer PWS1预处理）和70%乙醇。
操作步骤
使用前请先在Buffer PWS1中加入22 mL无水乙醇。
1. 向匀浆管A中加入250 mg土壤样本，确保样本均匀铺于管底。
2. 向匀浆管中加入750 µL Buffer MS1，涡旋振荡混匀15 sec，使土壤样本与缓冲液充分接触，初步分散样本。
3. 加入60 µL Buffer MS2，将离心管放入37℃、1500 rpm的恒温混匀仪上，振荡混匀10 min，充分发挥脱腐缓冲液的作用，去除土壤中的腐殖酸。
【注意】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。
4. 12,000 rpm (13,400 × g) 离心1 min，使杂质充分沉淀；小心转移上清液至新的2 mL离心管中。
5. 向上述离心管中加入250 µL Buffer MS3，充分混匀，涡旋振荡5 sec，置于4℃环境中放置5 min。
6. 12,000 rpm (13,400 × g) 离心1 min；转移上清液至新的2 mL离心管中，加入200 µL Buffer MS4，充分混匀，置于4℃环境中放置5 min。
7. 12,000 rpm (13,400 × g) 离心1 min，使杂质充分沉淀；转移上清液到新的2 mL离心管中，加入1200 μL Buffer MS5，颠倒混匀。
【注意】转移上清时不要移走沉淀，否则可能引入杂质，降低DNA纯度。
8. 将步骤7得到的溶液转入吸附柱PBF（将吸附柱放入配套的2 mL收集管中），12,000 rpm (13,400 × 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 PWS1（使用前已加入无水乙醇），12,000 rpm (13,400×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 70%乙醇（客户自备），12,000 rpm (13,400×g) 离心30 sec，弃去废液，将吸附柱放回收集管中，进一步去除残留盐分和杂质。
11. 将吸附柱连同收集管一起，12,000 rpm (13,400 × 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）。
12. 将吸附柱从收集管中取出，转入一个新的1.5 mL离心管中，室温开盖晾干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50-100 μL Buffer EB，室温放置1 min；12,000 rpm (13,400×g) 离心30 sec，收集离心管中的DNA溶液。
【注意】
(1) Buffer EB体积不应少于50 μL，体积过小会影响DNA回收效率。如用水做洗脱液，应保证其pH值在7.0-8.5范围内（可用NaOH将水的pH值调到此范围）。DNA溶液请于-20℃保存，防止降解。
(2) 将步骤12所得洗脱液重新加入吸附柱，室温放置2 min，12,000 rpm离心30 sec，可以提高DNA的终浓度。
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50 µg/mL双链DNA，或40 µg/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 uses a unique humus removal buffer system to maximally eliminate interfering impurities such as humic acid from soil samples. It is also equipped with homogenization tubes containing grinding beads that effectively disrupt various complex components in soil samples, ensuring the integrity of genomic DNA extracted from soil. 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, restriction enzyme digestion, and molecular markers.
| Product No. | Component | Appearance | 50T | Storage |
|---|
| S1522826A | Buffer MS1 | Liquid | 40 mL | RT | | S1522826B | Buffer MS2 | Liquid | 4 mL | RT | | S1522826C | Buffer MS3 | Liquid | 14 mL | RT | | S1522826D | Buffer MS4 | Liquid | 11 mL | RT | | S1522826E | Buffer MS5 | Liquid | 66 mL | RT | | S1522826F | Buffer PWS1 | Liquid | 17 mL | RT | | S1522826G | Buffer EB | Liquid | 15 mL | RT | | S1522826H | Homogenization Tube A | — | 1 EA × 50 | RT | | S1522826I | PBF Spin Column (with 2 mL collection tube) | — | 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 different soil types, consult the appropriate optimal storage conditions to avoid sample deterioration that may affect extraction results.
When pipetting supernatants, avoid sucking up any precipitate, as this may introduce impurities and 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 present, heat at 37°C until completely dissolved before use.
The user needs to supply absolute ethanol (for pretreatment of Buffer PWS1) and 70% ethanol.
Procedure
Before use, add 22 mL of absolute ethanol to Buffer PWS1.
1. Add 250 mg of soil sample to Homogenization Tube A, ensuring the sample is evenly distributed at the bottom of the tube.
2. Add 750 µL of Buffer MS1 to the tube. Vortex for 15 sec to thoroughly mix the soil sample with the buffer and initially disperse the sample.
3. Add 60 µL of Buffer MS2. Place the tube on a thermomixer at 37°C and 1500 rpm, and shake for 10 min to allow the humus removal buffer to effectively remove humic acid from the soil.
【Note】 Before use, check Buffer MS2 for precipitation. If present, heat at 37°C until completely dissolved before use.
4. Centrifuge at 12,000 rpm (13,400 × g) for 1 min to fully pellet impurities. Carefully transfer the supernatant to a new 2 mL centrifuge tube.
5. Add 250 µL of Buffer MS3 to the tube, mix thoroughly, vortex for 5 sec, and place at 4°C for 5 min.
6. Centrifuge at 12,000 rpm (13,400 × g) for 1 min. Transfer the supernatant to a new 2 mL centrifuge tube, add 200 µL of Buffer MS4, mix thoroughly, and place at 4°C for 5 min.
7. Centrifuge at 12,000 rpm (13,400 × g) for 1 min to fully pellet impurities. Transfer the supernatant to a new 2 mL centrifuge tube, add 1200 μL of Buffer MS5, and mix by inversion.
【Note】 Do not transfer any precipitate when removing the supernatant, as this may introduce impurities and reduce DNA purity.
8. Transfer the solution from step 7 into a PBF spin column (placed in the provided 2 mL collection tube). Centrifuge at 12,000 rpm (13,400 × 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 PWS1 (with ethanol pre‑added) to the spin column. Centrifuge at 12,000 rpm (13,400 × g) for 30 sec. Discard the flow‑through and return the spin column to the collection tube.
10. Add 700 μL of 70% ethanol (user‑supplied) to the spin column. Centrifuge at 12,000 rpm (13,400 × g) for 30 sec. Discard the flow‑through and return the spin column to the collection tube to further remove residual salts and impurities.
11. Centrifuge the spin column together with the collection tube at 12,000 rpm (13,400 × g) for 2 min to completely remove residual wash buffer and impurities (this avoids residual ethanol affecting subsequent DNA elution and experiments).
12. DNA elution
Transfer the spin column to a new 1.5 mL centrifuge tube. Open the cap and let stand at room temperature for 2 min to allow residual ethanol to evaporate completely. Slowly add 50–100 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 (13,400 × g) for 30 sec. Collect the DNA solution in the centrifuge tube.
【Notes】
(1) The volume of Buffer EB should not be less than 50 μL, as too small a volume will reduce DNA recovery efficiency. If water is used for elution, ensure its pH is between 7.0 and 8.5 (adjust with NaOH). Store the DNA solution at –20°C to prevent degradation.
(2) Reloading the eluate from step 12 onto the spin column, letting it stand at room temperature for 2 min, and centrifuging at 12,000 rpm for 30 sec can increase the final DNA concentration.
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 (A260) 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 A260/A280 ratio. For high‑quality genomic DNA, the A260/A280 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.
ZJ26F0838268
危险品化学品经营许可证（带存储）