本试剂盒采用可特异结合DNA的离心吸附柱和独特的缓冲液体系，可快速提取真菌的基因组DNA，特别适用于霉菌、酵母菌以及蕈菌的基因组DNA提取。该试剂盒提取得率高、稳定性好，使用本试剂盒回收的DNA可适用于酶切、PCR和文库构建等各种常规实验操作。
产品号
| 组件
| 物理外观
| 50T
| 储存温度
|
F1522818A
| Buffer MPL
| 液体
| 22 mL | RT.
|
F1522818B
| Buffer MPB
| 液体
| 30 mL | RT.
|
F1522818C
| Buffer MPW
| 液体
| 38 mL | RT.
|
F1522818D
| Buffer PW2
| 液体
| 15 mL | RT.
|
F1522818E
| Buffer EB
| 液体
| 15 mL
| RT.
|
F1522818F
| 吸附柱WHF (含2mL收集管)
| —
| 1 EA×50
| RT.
|
F1522818G
| 匀浆管D
| —
| 1 EA×50
| RT. |
本试剂盒所有组分可置于室温干燥条件下保存15个月。
注意事项
1. 样品应避免反复冻融，否则会导致提取的DNA片段较小且提取量下降。
2. 实验需自备无水乙醇、异丙醇、RNase A (100 mg/mL)、离心管，蕈菌提取需额外准备液氮。
操作步骤
使用前请先在Buffer PW2中加入60 mL的无水乙醇。
1. 样本前处理
1.1 酵母菌
将培养至OD值为1.0-5.0的酵母培养液1-2 mL（最多不超过5 mL）加入匀浆管D中，12,000 rpm (13,680 × g) 离心2 min，弃去上清液，保留菌体沉淀；向沉淀中加入400 μL Buffer MPL进行匀质处理。将匀浆管固定在装有2 mL适配器的振荡研磨装置中，根据设备型号使用优化的研磨条件进行处理（具体方法参见附录）。
1.2 蕈菌
将真菌菌体进行液氮研磨，在低温条件下称取不超过100 mg研磨后的样品，转移到2 mL离心管中，加入400 μL Buffer MPL，充分涡旋（或均质20 sec），直至溶液无明显结块，确保样本充分分散。
1.3 霉菌
挑取真菌菌丝或将培养的菌体（不超过100 mg）加入匀浆管D中，加入400 μL Buffer MPL进行均质处理（研磨程序同酵母菌）；也可按照蕈菌的液氮研磨步骤进行处理。
2. 向上述处理后的样本中加入4 μL RNase A (100 mg/mL) 溶液，涡旋振荡15 sec，室温放置5 min，去除RNA杂质；然后将匀浆管放入70℃金属浴或水浴中孵育10 min，孵育过程中颠倒混合样品数次，促进菌体充分裂解。
3. 14,000 rpm (18,630 × g) 离心4 min，使杂质充分沉淀；小心转移上清液至新的1.5 mL离心管中，避免吸入溶液底部的沉淀。
4. 向离心管中加入550 µL Buffer MPB，充分颠倒混匀，此时可能会出现絮状沉淀，属于正常现象；将得到的溶液和沉淀一起转入吸附柱WHF（将吸附柱放入配套的2 mL收集管中），每次上样量不超过700 μL（如果混合液体积超过700 μL，需要分两次转入）；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。
5. 向吸附柱中加入700 μL Buffer MPW，12,000 rpm (13,680×g) 离心30 sec，弃去废液，将吸附柱放回收集管中。
6. 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。
7. 重复步骤6（第二次洗涤）的操作，进一步去除吸附柱上残留的盐分、蛋白质等杂质，提升DNA纯度。
8. 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）。
9. 将吸附柱从收集管中取出，转入一个新的1.5 mL离心管中，开盖晾干2 min，彻底挥发吸附柱中残留的乙醇。向吸附膜的中间部位缓慢悬空滴加30-100 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。
【注意】
(1) Buffer EB体积不应小于30 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9所得洗脱液重新加入吸附柱，12,000 rpm (13,680×g) 离心30 sec，可以提高DNA的终浓度。
(3) 用另外的30-100 μL Buffer EB再次洗脱，可以增加DNA的提取产量。
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 uses a spin column that specifically binds DNA and a unique buffer system to rapidly extract genomic DNA from fungi. It is particularly suitable for the extraction of genomic DNA from molds, yeasts, and mushrooms. The kit offers high yield and good stability. The DNA recovered with this kit can be used in various routine experimental procedures such as restriction enzyme digestion, PCR, and library construction.
| Product No. | Component | Appearance | 50T | Storage |
|---|
| F1522818A | Buffer MPL | Liquid | 22 mL | RT | | F1522818B | Buffer MPB | Liquid | 30 mL | RT | | F1522818C | Buffer MPW | Liquid | 38 mL | RT | | F1522818D | Buffer PW2 | Liquid | 15 mL | RT | | F1522818E | Buffer EB | Liquid | 15 mL | RT | | F1522818F | WHF Spin Column (with 2 mL collection tube) | — | 1 EA × 50 | RT | | F1522818G | Homogenization Tube D | — | 1 EA × 50 | RT |
|
All components of this kit can be stored under dry conditions at room temperature for 15 months.
Precautions
Samples should be protected from repeated freeze‑thaw cycles, which may result in smaller DNA fragments and reduced yield.
The user needs to supply absolute ethanol, isopropanol, RNase A (100 mg/mL), centrifuge tubes. For mushroom extraction, liquid nitrogen is also required.
Procedure
Before use, add 60 mL of absolute ethanol to Buffer PW2.
1. Sample Pretreatment
1.1 Yeast
Transfer 1–2 mL (maximum 5 mL) of yeast culture with an OD value of 1.0–5.0 into Homogenization Tube D. Centrifuge at 12,000 rpm (13,680 × g) for 2 min. Discard the supernatant and retain the cell pellet. Add 400 μL of Buffer MPL to the pellet for homogenization. Secure the homogenization tube in a bead‑beating device with 2‑mL adapters and process using optimized conditions according to the device (see Appendix for details).
1.2 Mushroom
Grind the fungal tissue with liquid nitrogen. Weigh no more than 100 mg of the ground sample under low‑temperature conditions and transfer to a 2 mL centrifuge tube. Add 400 μL of Buffer MPL, vortex thoroughly (or homogenize for 20 sec) until the solution has no obvious clumps, ensuring complete sample dispersion.
1.3 Mold
Transfer fungal mycelium or cultured tissue (no more than 100 mg) into Homogenization Tube D. Add 400 μL of Buffer MPL and homogenize (grinding procedure same as for yeast). Alternatively, process following the liquid nitrogen grinding procedure for mushrooms.
2. Add 4 μL of RNase A solution (100 mg/mL) to the processed sample from step 1. Vortex for 15 sec and let stand at room temperature for 5 min to remove RNA impurities. Then place the homogenization tube in a 70°C metal bath or water bath and incubate for 10 min. During incubation, invert the tube several times to mix and promote complete cell lysis.
3. Centrifuge at 14,000 rpm (18,630 × g) for 4 min to fully pellet impurities. Carefully transfer the supernatant to a new 1.5 mL centrifuge tube, avoiding any precipitate at the bottom.
4. Add 550 µL of Buffer MPB to the tube and mix thoroughly by inversion. Flocculent precipitate may appear; this is normal. Transfer the solution and precipitate together into a WHF spin column (placed in the provided 2 mL collection tube). Load no more than 700 μL per transfer (if the mixture volume exceeds 700 μL, transfer in two portions). Centrifuge at 12,000 rpm (13,680 × g) for 30 sec. Discard the flow‑through and return the spin column to the collection tube.
5. Add 700 μL of Buffer MPW 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.
6. 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.
7. Repeat step 6 (second wash) to further remove residual salts, proteins, and other impurities on the spin column, thereby increasing DNA purity.
8. 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).
9. 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 30–100 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 (13,680 × g) for 30 sec. Collect the DNA solution in the centrifuge tube.
【Notes】
(1) The volume of Buffer EB should not be less than 30 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 9 onto the spin column and centrifuging at 12,000 rpm (13,680 × g) for 30 sec can increase the final DNA concentration.
(3) A second elution with another 30–100 μL of Buffer EB can increase the DNA yield.
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.
ZJ26F0636851
危险品化学品经营许可证（带存储）