本试剂盒适合从酵母细胞中提取基因组DNA，提取的基因组DNA片段大、纯度高、稳定性好，可直接用于PCR、酶切和杂交等实验。提取过程无需酚-氯仿抽提，酵母细胞样品经裂解液裂解和蛋白酶K消化后，在无水乙醇的调节下，基因组DNA可与吸附柱WHF特异性结合；经过快速充分的洗涤步骤，可有效去除残留的蛋白质、盐分等杂质；最后，DNA溶解于Buffer EB中，获得高纯度的基因组DNA。
产品号
| 组件
| 物理外观
| 50T
| 储存温度
|
Y1522816A
| Buffer DYE
| 液体
| 28 mL | RT.
|
Y1522816B
| Buffer UA
| 液体
| 15 mL | RT.
|
Y1522816C
| Buffer UL
| 液体
| 15 mL
| RT.
|
Y1522816D
| Buffer PW1
| 液体
| 17 mL | RT.
|
Y1522816E
| Buffer PW2
| 液体
| 15 mL
| RT.
|
Y1522816F
| Buffer EB
| 液体
| 15 mL
| RT.
|
Y1522816G
| 蛋白酶K
| 液体
| 1.2 mL
| RT.
|
Y1522816H
| 吸附柱WHF (含2mL收集管)
| —
| 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（用于Buffer PW1和Buffer PW2预处理）、Lyticase；如需去除RNA，需额外自备RNase A (100 mg/mL)、离心管。
3. 使用前请检查Buffer UA是否出现结晶或者沉淀，如有结晶或者沉淀，请将Buffer UA置于37℃水浴中重新溶解后使用。
操作步骤
使用前请先在Buffer PW1中加入22 mL无水乙醇，Buffer PW2中加入60 mL无水乙醇。
本方案适合于从<5×10⁷酵母细胞中提取高纯度基因组DNA。
1. 取酵母细胞（最多不超过5×10⁷个细胞），12,000 rpm (13,680×g) 离心1 min，尽量吸除上清液；若菌液较多，可通过多次离心将菌体沉淀收集到一个离心管中，减少试剂浪费。
2. 用500 μL Buffer DYE彻底重悬菌体，加入2 µL Lyticase，37℃孵育15 min，充分破坏酵母细胞壁；4,000 rpm (1,500×g) 离心10 min，弃去上清液，收集菌体沉淀。
3. 向菌体沉淀中加入200 µL Buffer UA和20 µL 蛋白酶K，涡旋振荡，使沉淀彻底重悬。
【注意】如果需要去除RNA，可加入4 μL RNase A (100 mg/mL) 溶液（客户自备），振荡15 sec，室温放置5 min，去除RNA杂质。
4. 加入220 µL Buffer UL，充分颠倒混匀，70℃放置10 min，此时溶液应变清亮；简短离心，去除管盖内壁的水珠，避免试剂残留。
【注意】加入Buffer UL时可能会产生白色沉淀，一般70℃放置时会消失，不会影响后续实验。
5. 加入220 µL无水乙醇，充分颠倒混匀，此时可能会出现絮状沉淀，属于正常现象；简短离心，去除管盖内壁的水珠，避免试剂残留。
6. 将步骤5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），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。
7. 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。
8. 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。
9. 重复步骤8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。
10. 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）。
11. 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50-100 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11所得洗脱液重新加至吸附柱上，室温放置2 min，12,000 rpm (13,680×g) 离心30 sec，可以提高DNA的终浓度。
(3) 用另外的50-100 μL Buffer EB再次洗脱，可以增加DNA的提取产量。
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 is suitable for extracting genomic DNA from yeast cells. The extracted genomic DNA has large fragments, high purity, and good stability, and can be directly used in experiments such as PCR, restriction enzyme digestion, and hybridization. The extraction process does not require phenol‑chloroform extraction. Yeast cells are lysed with a lysis buffer and digested with proteinase K. Then, in the presence of absolute ethanol, the genomic DNA binds specifically to the WHF spin column. After rapid and thorough washing steps, residual proteins, salts, and other impurities are effectively removed. Finally, the DNA is dissolved in Buffer EB, yielding high‑purity genomic DNA.
| Product No. | Component | Appearance | 50T | Storage |
|---|
| Y1522816A | Buffer DYE | Liquid | 28 mL | RT | | Y1522816B | Buffer UA | Liquid | 15 mL | RT | | Y1522816C | Buffer UL | Liquid | 15 mL | RT | | Y1522816D | Buffer PW1 | Liquid | 17 mL | RT | | Y1522816E | Buffer PW2 | Liquid | 15 mL | RT | | Y1522816F | Buffer EB | Liquid | 15 mL | RT | | Y1522816G | Proteinase K | Liquid | 1.2 mL | RT | | Y1522816H | WHF Spin Column (with 2 mL collection tube) | — | 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 (for pretreatment of Buffer PW1 and Buffer PW2), Lyticase. If RNA removal is required, RNase A solution (100 mg/mL) and centrifuge tubes are also needed.
Before use, check Buffer UA for crystallization or precipitation. If present, redissolve by placing in a 37°C water bath.
Procedure
Before use, add 22 mL of absolute ethanol to Buffer PW1 and 60 mL of absolute ethanol to Buffer PW2.
This protocol is suitable for extracting high‑purity genomic DNA from <5×10⁷ yeast cells.
1. Collect yeast cells (maximum 5×10⁷ cells). Centrifuge at 12,000 rpm (13,680 × g) for 1 min and remove the supernatant as completely as possible. If the culture volume is large, the cell pellet can be collected into a single tube by multiple centrifugations to minimize reagent waste.
2. Thoroughly resuspend the cell pellet in 500 μL of Buffer DYE. Add 2 µL of Lyticase and incubate at 37°C for 15 min to fully disrupt the yeast cell wall. Centrifuge at 4,000 rpm (1,500 × g) for 10 min, discard the supernatant, and collect the cell pellet.
3. Add 200 µL of Buffer UA and 20 µL of Proteinase K to the cell pellet. Vortex to completely resuspend the pellet.
【Note】 If RNA removal is required, add 4 μL of RNase A solution (100 mg/mL) (user‑supplied), vortex for 15 sec, and let stand at room temperature for 5 min to remove RNA impurities.
4. Add 220 µL of Buffer UL and mix thoroughly by inversion. Incubate at 70°C for 10 min; the solution should become clear. Briefly centrifuge to collect drops from the cap wall, avoiding reagent residue.
【Note】 White precipitate may form upon addition of Buffer UL, but usually disappears during incubation at 70°C and does not affect subsequent steps.
5. Add 220 µL of absolute ethanol and mix thoroughly by inversion. Flocculent precipitate may appear; this is normal. Briefly centrifuge to collect drops from the cap wall, avoiding reagent residue.
6. Transfer the solution and precipitate from step 5 into a WHF 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.
7. 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.
8. 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.
9. Repeat step 8 to further remove residual salts, proteins, and other impurities on the spin column, thereby increasing DNA purity.
10. 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).
11. 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 50–100 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 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 11 onto the spin column, letting it stand at room temperature for 2 min, and centrifuging at 12,000 rpm (13,680 × g) for 30 sec can increase the final DNA concentration.
(3) A second elution with another 50–100 μL of Buffer EB can increase the DNA yield.
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.
ZJ26F0636936
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