本试剂盒适合从海洋动物组织样品中快速简单地提取基因组DNA，提取的基因组DNA片段大，纯度高，稳定性好，可直接用于PCR、酶切和杂交等实验。提取过程无需酚氯仿抽提，血液、细胞或组织经裂解液裂解和蛋白酶K消化，然后在无水乙醇的调节下可与吸附柱WHF结合，经过快速充分的洗涤去除残留的蛋白质和盐分等杂质，最后DNA溶解于Buffer EB中。
产品号
| 组件
| 物理外观
| 50T
| 储存温度
|
M1522830A
| Buffer UA
| 液体
| 15 mL | RT.
|
M1522830B
| Buffer UL
| 液体
| 15 mL
| RT.
|
M1522830C
| Buffer PW1
| 液体
| 17 mL | RT.
|
M1522830D
| Buffer PW3
| 液体
| 15 mL
| RT.
|
M1522830E
| Buffer EB
| 液体
| 15 mL
| RT.
|
M1522830F
| 蛋白酶K
| 液体
| 1.2 mL | RT.
|
M1522830G
| 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 UA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
1. 样品应避免反复冻融，否则会导致提取的DNA片段较小且提取量也下降。
2. 如需要去除RNA，需自备试剂RNase A (100 mg/mL)。
3. 自备无水乙醇。
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. 样本处理
切取不超过30 mg的海洋动物组织材料，放入装有200 μL Buffer UA的离心管中，涡旋振荡15 sec。【注意】根据提取的组织不同，起始量也稍有不同；腮组织的细胞量较大，一般建议提取量不超过20 mg。若需要去除RNA，可加入4 μL RNase A (100 mg/mL) 溶液，涡旋振荡混匀15 sec，室温放置5-10 min。
2. 加入20 μL 蛋白酶K，涡旋振荡混匀。56℃孵育直至组织完全溶解，短暂离心以收集管盖内壁上的溶液，即可进行下一步。
【注意】不同海洋动物组织的裂解时间不同，通常需0.5–2 h即可完成；其中扇贝组织0.5 h基本可裂解完全，虾和鱼类组织约需1 h。孵育过程中，每小时振荡混合样品2-3次，每次振荡混匀15 sec，以促进组织裂解。
3. 加入200 μL Buffer UL，充分颠倒混匀，70℃孵育10 min，短暂离心以收集管盖内壁上的溶液。【注意】加入Buffer UL时可能会产生白色沉淀，一般在70℃孵育过程中会自行消失，不会影响后续实验。
4. 加入200 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）；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2mL收集管中），12,000 rpm（13,680 × g）离心30sec，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700μL Buffer PW1（使用前已加入无水乙醇），12,000 rpm（13,680 × g）离心30sec，弃去废液，将吸附柱放回收集管中。
6. 向吸附柱中加入700μL Buffer PW3（使用前已加入无水乙醇），12,000 rpm（13,680 × g）离心30sec，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50-200 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) 若下游实验对pH值或EDTA敏感，可改用灭菌水进行洗脱。洗脱液的pH值对洗脱效率影响较大，若用水作为洗脱液，需保证其pH值在7.0-8.5之间（可使用NaOH调节），pH值低于7.0会导致洗脱效率下降。
(2) 若需提高DNA的终浓度，可将步骤9收集到的DNA洗脱液重新加至同一吸附膜上，室温放置2 min，12,000 rpm（13,680 × g）离心30 sec，再次收集洗脱液即可。
(3) 若需长期保存DNA，建议使用Buffer EB进行洗脱，并将DNA溶液置于-20℃保存，可有效防止DNA降解。
DNA浓度及纯度检测
提取的基因组DNA片段大小，受样品保存时间、操作过程中的剪切力等因素影响。纯化后的DNA片段可通过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，若用水稀释，可能导致检测数值不准确。260 nm处的吸光度应控制在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。若洗脱时未使用Buffer EB，而是使用ddH₂O，可能会导致比值偏低，这是由于pH值和离子存在影响光吸收值所致，并不代表DNA纯度不足。
This kit is suitable for rapid and simple extraction of genomic DNA from marine animal tissue samples. 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. Blood, cells, or tissues are lysed with a lysis buffer and digested with proteinase K, then in the presence of absolute ethanol the DNA binds to the WHF spin column. After thorough washing to remove residual proteins, salts, and other impurities, the DNA is finally eluted with Buffer EB.
| Product No. | Component | Appearance | 50T | Storage |
|---|
| M1522830A | Buffer UA | Liquid | 15 mL | RT | | M1522830B | Buffer UL | Liquid | 15 mL | RT | | M1522830C | Buffer PW1 | Liquid | 17 mL | RT | | M1522830D | Buffer PW3 | Liquid | 15 mL | RT | | M1522830E | Buffer EB | Liquid | 15 mL | RT | | M1522830F | Proteinase K | Liquid | 1.2 mL | RT | | M1522830G | 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 UA 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.
If RNA removal is required, the user needs to supply RNase A solution (100 mg/mL).
The user needs to supply absolute ethanol.
Procedure
Before use, add 22 mL of absolute ethanol to Buffer PW1 and 60 mL of absolute ethanol to Buffer PW3.
1. Sample Processing
Cut no more than 30 mg of marine animal tissue and place into a centrifuge tube containing 200 μL of Buffer UA. Vortex for 15 sec.
【Note】 The starting amount varies slightly depending on the tissue type. Gill tissue contains a high cell mass; generally, it is recommended not to exceed 20 mg. If RNA removal is required, add 4 μL of RNase A solution (100 mg/mL), vortex for 15 sec, and let stand at room temperature for 5–10 min.
2. Add 20 μL of Proteinase K and vortex to mix. Incubate at 56°C until the tissue is completely dissolved. Briefly centrifuge to collect the solution from the cap wall, then proceed to the next step.
【Note】 The lysis time varies among different marine animal tissues, typically 0.5–2 h. Scallop tissue usually lyses completely within 0.5 h; shrimp and fish tissues take about 1 h. During incubation, mix the sample by vortexing for 15 sec 2–3 times per hour to promote tissue lysis.
3. Add 200 μL of Buffer UL and mix thoroughly by inversion. Incubate at 70°C for 10 min. Briefly centrifuge to collect the solution from the cap wall.
【Note】 White precipitate may form upon addition of Buffer UL, but it usually disappears during incubation at 70°C and does not affect subsequent steps.
4. Add 200 μL of absolute ethanol 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). 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 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.
6. Add 700 μL of Buffer PW3 (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 impurities on the spin column and increase 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 50–200 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 instead. 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 9 onto the same membrane, let stand at room temperature for 2 min, centrifuge at 12,000 rpm (13,680 × g) for 30 sec, and collect the eluate again.
(3) For long‑term DNA storage, it is recommended to elute with Buffer EB and store the DNA solution at –20°C to effectively prevent DNA degradation.
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.
ZJ26F0636883
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