本试剂盒适合从口腔拭子、咽拭子以及漱口水（唾液）等多种口腔样本中快速、简便地提取基因组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、核酸杂交等实验。提取过程无需酚-氯仿抽提，口腔样本中的细胞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
|
O1522835A
| Buffer MUA
| 液体
| 30 mL | RT.
|
O1522835B
| Buffer DSL
| 液体
| 15 mL
| RT.
|
O1522835C
| Buffer RW1
| 液体
| 15 mL
| RT.
|
O1522835D
| Buffer PW2
| 液体
| 15 mL
| RT.
|
O1522835E
| Buffer EB
| 液体
| 15 mL
| RT.
|
O1522835F
| 蛋白酶K
| 液体
| 0.7 mL | RT.
|
O1522835G
| 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。
使用前请检查Buffer MUA是否出现结晶或者沉淀，如有结晶或者沉淀，请置于37℃水浴重新溶解。
注意事项
- 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。
- 实验需自备无水乙醇和异丙醇，分别用于Buffer RW1、Buffer PW2和Buffer DSL的预处理。
操作步骤
使用前请先在Buffer RW1中加入23 mL无水乙醇，Buffer PW2中加入60 mL的无水乙醇，Buffer DSL中加入15 mL异丙醇。
1. 样本处理（按样本类型选择对应方法）
1.1 口腔拭子样本
将在面颊内侧擦拭过的拭子转移到2 mL离心管中，加入500 μL Buffer MUA和10 μL 蛋白酶K，涡旋振荡混匀10 sec；65℃放置15-30 min，每隔10 min涡旋混匀数次；取出300-350 μL处理后的样品，进行后续步骤。
1.2 咽拭子样本
将在咽部擦拭过的拭子转移到5 mL离心管中，加入1-2 mL的Buffer MUA，颠倒混匀；取300-350 μL含有Buffer MUA的咽拭子样本，加入10 μL 蛋白酶K，涡旋振荡混匀10 sec；65℃放置30 min，每隔10 min涡旋混匀数次，进行后续步骤。
1.3 唾液（漱口水）样本
在唾液样本中加入等体积的Buffer MUA，颠倒混匀；取300-350 μL含有Buffer MUA的唾液样本，加入10 μL 蛋白酶K，涡旋振荡混匀10 sec；65℃放置30 min，每隔10 min涡旋混匀数次，进行后续步骤。【注意】如果样本已保存在其他厂家的样品保存液中，需加入样品1/2体积的Buffer MUA和10 μL 蛋白酶K，涡旋振荡混匀10 sec；65℃放置15-30 min，取出300-350 μL处理后的样品，进行后续步骤。
2. 向步骤1处理后的300-350 μL样品中，加入500 μL Buffer DSL（使用前已加入异丙醇），充分颠倒混匀，室温放置5 min。
3. 将步骤2得到的溶液转入吸附柱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。
4. 向吸附柱中加入700 μL Buffer RW1（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。
5. 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。
6. 重复步骤5（第二次洗涤）的操作，进一步去除吸附柱上残留的杂质，提升DNA纯度。
7. 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）。
8. 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。
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 rapid and simple extraction of genomic DNA from various oral samples, including buccal swabs, throat swabs, and mouthwash (saliva). 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 nucleic acid hybridization. The extraction process does not require phenol‑chloroform extraction. Cells in the oral sample 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 |
|---|
| O1522835A | Buffer MUA | Liquid | 30 mL | RT | | O1522835B | Buffer DSL | Liquid | 15 mL | RT | | O1522835C | Buffer RW1 | Liquid | 15 mL | RT | | O1522835D | Buffer PW2 | Liquid | 15 mL | RT | | O1522835E | Buffer EB | Liquid | 15 mL | RT | | O1522835F | Proteinase K | Liquid | 0.7 mL | RT | | O1522835G | 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.
Before use, check Buffer MUA for crystallization or precipitation. If present, redissolve by placing in a 37°C water bath.
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 and isopropanol for pretreatment of Buffer RW1, Buffer PW2, and Buffer DSL.
Procedure
Before use, add 23 mL of absolute ethanol to Buffer RW1, 60 mL of absolute ethanol to Buffer PW2, and 15 mL of isopropanol to Buffer DSL.
1. Sample Processing (choose according to sample type)
1.1 Buccal swab samples
Transfer the swab that has been rubbed on the inside of the cheek into a 2 mL centrifuge tube. Add 500 μL of Buffer MUA and 10 μL of Proteinase K. Vortex for 10 sec. Incubate at 65°C for 15–30 min, vortexing several times every 10 min. Take 300–350 μL of the processed sample and proceed to the next step.
1.2 Throat swab samples
Transfer the swab that has been rubbed on the throat into a 5 mL centrifuge tube. Add 1–2 mL of Buffer MUA and mix by inversion. Take 300–350 μL of the throat swab sample containing Buffer MUA, add 10 μL of Proteinase K, and vortex for 10 sec. Incubate at 65°C for 30 min, vortexing several times every 10 min. Proceed to the next step.
1.3 Saliva (mouthwash) samples
Add an equal volume of Buffer MUA to the saliva sample and mix by inversion. Take 300–350 μL of the saliva sample containing Buffer MUA, add 10 μL of Proteinase K, and vortex for 10 sec. Incubate at 65°C for 30 min, vortexing several times every 10 min. Proceed to the next step.
【Note】 If the sample has been stored in a sample preservation solution from another manufacturer, add half the sample volume of Buffer MUA and 10 μL of Proteinase K. Vortex for 10 sec. Incubate at 65°C for 15–30 min. Take 300–350 μL of the processed sample and proceed.
2. To the 300–350 μL of processed sample from step 1, add 500 μL of Buffer DSL (with isopropanol pre‑added). Mix thoroughly by inversion and let stand at room temperature for 5 min.
3. Transfer the solution from step 2 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.
4. Add 700 μL of Buffer RW1 (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.
5. 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.
6. Repeat step 5 (second wash) to further remove residual impurities on the spin column, thereby increasing DNA purity.
7. 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).
8. 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.
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.
ZJ26F0636884
危险品化学品经营许可证（带存储）