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本试剂盒适合从各种血液/细胞/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 Proteinase K 消化，然后在无水乙醇的调节下可与 Spin Columns GH4 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。
产品组分及储存条件：
| M665559 | Component | 50T | Storage |
| M665559A | Buffer DA | 15 ml | RT |
| M665559B | Buffer DLB | 15 ml | RT |
| M665559C | Buffer DW1 | 13 ml | RT |
| M665559D | Buffer DW2Plus | 12 ml | RT |
| M665559E | Buffer EB | 15 ml | RT |
| M665559F | Proteinase K（20 mg/ml） | 1.2 ml | RT |
| M665559G | Spin Columns GH4 | 50 pcs | RT |
| M665559H | 2 ml Collection Tubes | 50 pcs | RT |
储存条件：
本试剂盒所有组分可置于室温15°C-25°C干燥条件下保存 12 个月。使用前请检查 Buffer DA 和 Buffer DLB 是否出现结晶或者沉淀，如有结晶或者沉淀，请将 Buffer DA 和 Buffer DLB 置于 37°C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. 首次使用前，Buffer DW1 (13mL) 需添加 17mL 无水乙醇，Buffer DW2Plus (12mL) 需添加 48mL 无水乙醇。
3. 如需要去除 RNA，需自备试剂 RNase A（100 mg/ml）（目录号：R665518）。
4. 自备无水乙醇试剂。
操作步骤：
使用前请先在 Buffer DW1 和 Buffer DW2Plus 中加入无水乙醇，加入体积请参照瓶上的标签。
1. 样本处理：
a. 哺乳动物血液：取 200 μl 新鲜或抗凝血液放入 1.5 ml 离心管中，直接按照步骤 2-10 进行操作，当血液不足 200 μl 时需用 Buffer DA 补足。
b. 禽类、鸟类、两栖类血液：取 5-20 μl 新鲜或抗凝血液放入 1.5 ml 离心管中，用 Buffer DA 补足 200 μl。
c. 组织培养细胞：收集约 10⁵-10⁶ 个悬浮细胞（细胞总量不可超过 5×10⁶ 个）到一个 1.5 ml 离心管；对于贴壁细胞，应先用胰蛋白酶消化吹打下来。然后 12,000 rpm（~13,400×g）离心 1 min，弃上清，向细胞沉淀中加入 200 μl Buffer DA，振荡至彻底悬浮。
d. 动物组织：称取< 25 mg（脾脏用量应少于 10 mg）动物组织经液氮研磨成细粉或用解剖刀切成小碎块，转入预先装有 180 μl Buffer DA 的 1.5 ml 离心管。
本试剂盒除了较好提取血液、细胞和组织 DNA 外，还能从细菌中提取高质量的 DNA。
e. 细菌：取细菌培养液 1-5 ml，12,000 rpm（~13,400×g）离心 1 min，尽量吸净上清。
1) 对于较难破壁的革兰氏阳性菌，加入溶菌酶溶液进行破壁处理，具体方法为：加入 110 μl 缓冲液（20 mM Tris, pH 8.0；2 mM Na₂-EDTA；1.2% Triton）和 70 μl 溶菌酶溶液（50 mg/ml，客户自备），37°C处理 30 min 以上。
2) 对于相对容易裂解的细菌，可直接向菌体沉淀中加入 200 μl Buffer DA，振荡至菌体彻底悬浮。
2. 可选步骤：如果需要去除 RNA（细胞、组织和细菌需去除 RNA），可加入 4 μl RNase A（100 mg/ml）溶液（目录号：R665518）。振荡 15 sec，室温放置 5 min。
3. 加入 20 μl Proteinase K，涡旋振荡混匀。
a. 如提取样本为血液、细胞或细菌，只需加入 Proteinase K 充分混匀，即可继续进行下一步。
b. 如提取样本为动物组织，加入 Proteinase K，涡旋振荡混匀。56°C 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：不同组织裂解时间不同，通常需 1–3 h 即可完成（鼠尾需要消化 6-8 h，必要时消化过夜），不会影响后续操作。
4. 加入 200 μl Buffer DLB，充分颠倒混匀，70°C 孵育 10 min，短暂离心以收集管盖内壁上的溶液。
注意：加入 Buffer DLB 时可能会产生白色沉淀，一般 70°C 孵育时会消失，不会影响后续实验。
5. 加入 200 μl 无水乙醇，混匀（此时可能会出现沉淀），将得到的溶液和沉淀一起转入吸附柱 Spin Columns GH4（吸附柱放在收集管中），12,000 rpm（~13,400×g）离心 30 sec，弃去滤液，将吸附柱放回收集管中。
6. 加入 500 μl Buffer DW1（使用前请加入无水乙醇）至吸附柱，12,000 rpm（~13,400×g）离心 30 sec，弃废液，将吸附柱放回收集管中。
7. 加入 500 μl Buffer DW2Plus（使用前请加入无水乙醇）至吸附柱，12,000 rpm（~13,400×g）离心 30 sec，弃废液，将吸附柱放回收集管中。
8. 重复操作步骤 7 一次。
9. 12,000 rpm 离心 3 min，倒掉收集管中的废液。
10. 将吸附柱转入一个新的 1.5 ml 离心管中，向吸附膜的中间部位悬空滴加 60-200 μl Buffer EB，室温放置 1 min，12,000 rpm（~13,400×g）离心 1 min，得到 DNA 溶液。
注意：Buffer EB 体积不应少于 60 μl，体积过小影响回收效率。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°C保存。
常见问题及解决方案：
| 常见问题 | 原因 | 解决方案 |
| 柱子堵塞 | 1. 样本投入量太多 | 请按照兼容范围投入样本。 |
| 2. 样本裂解不充分 | 延长 56°C水浴时间，增加颠倒混匀次数。 | |
| DNA 产量低 | 1. 样本被反复冻融 | 避免反复冻融样本，推荐使用新鲜样本或只冻融过一次的样本。 |
| 2. 动物组织裂解不充分 | 组织块尽量剪碎或液氮研磨，提取时使样本及时与 Buffer DA 及 Proteinase K充分混匀，且可适当延长 56°C水浴裂解时间。 | |
| 3. 未完全将裂解混合物转移至吸附柱内 | 细胞裂解后加无水乙醇，溶液中会出现絮状沉淀，需要将其同溶液一起转移至吸附柱内。 | |
| 4. 洗脱液问题 | 请使用试剂盒自带的 Buffer EB 洗脱，若用 ddH2O 或其他洗脱液，确认洗脱液pH 值在 7.0 - 8.0 之间。可提前将 Buffer EB 预热至 55°C后再进行洗脱，有利于提高 DNA 得率。 | |
| 5. 洗脱效率低 | 洗脱液需滴加于膜中央位置；增加洗脱体积或洗脱次数。 | |
| 6. Buffer DW1/DW2Plus未添加无水乙醇 | 按瓶身标签所示，加入指定体积的无水乙醇至 Buffer DW1 和 Buffer DW2Plus中。 | |
| DNA 纯度低 | 1. 杂蛋白污染 | 未使用 Buffer DW1 漂洗，或 Buffer DW1 中未加入正确体积的无水乙醇。请按瓶身标签加入指定体积的无水乙醇后操作。 |
| 2. 杂质离子污染 | 未使用 Buffer DW2Plus 漂洗，或只漂洗一次。请按照说明书使用 Buffer DW2Plus 漂洗两次。 |
This kit is suitable for rapid and simple extraction of genomic DNA from various blood/cell/tissue samples. The extracted genomic DNA features large fragment size, high purity, and good stability, and can be directly used in experiments such as PCR, enzyme digestion, and hybridization. The extraction process does not require phenol-chloroform extraction: blood, cells, or tissues are lysed with lysis buffer and digested with Proteinase K, then can bind to Spin Columns GH4 under the adjustment of absolute ethanol. Residual impurities such as proteins and salts are removed through rapid and thorough washing, and finally, DNA is eluted in Buffer EB.
Product Components and Storage Conditions:
| M665559 | Component | 50T | Storage |
| M665559A | Buffer DA | 15 ml | RT |
| M665559B | Buffer DLB | 15 ml | RT |
| M665559C | Buffer DW1 | 13 ml | RT |
| M665559D | Buffer DW2Plus | 12 ml | RT |
| M665559E | Buffer EB | 15 ml | RT |
| M665559F | Proteinase K（20 mg/ml） | 1.2 ml | RT |
| M665559G | Spin Columns GH4 | 50 pcs | RT |
| M665559H | 2 ml Collection Tubes | 50 pcs | RT |
Storage Conditions:
All components of this kit can be stored at room temperature (15°C-25°C) under dry conditions for 12 months. Before use, check if Buffer DA and Buffer DLB have crystals or precipitates. If crystals or precipitates are present, place Buffer DA and Buffer DLB in a 37°C water bath to redissolve.
Precautions:
1. Samples should avoid repeated freezing and thawing, as this will result in smaller extracted DNA fragments and reduced extraction yield.
2. Before first use, 17mL of absolute ethanol must be added to Buffer DW1 (13mL), and 48mL of absolute ethanol must be added to Buffer DW2Plus (12mL).
3. If RNA removal is required, RNase A (100 mg/ml) (Catalog No.: R665518) must be prepared separately.
4. Absolute ethanol must be prepared separately.
Operating Steps:
Before use, add absolute ethanol to Buffer DW1 and Buffer DW2Plus. Please refer to the labels on the bottles for the volume to be added.
1. Sample Processing:
a. Mammalian blood: Take 200 μl of fresh or anticoagulated blood into a 1.5 ml centrifuge tube, and proceed directly to steps 2-10. If the blood volume is less than 200 μl, make up to 200 μl with Buffer DA.
b. Avian, poultry, and amphibian blood: Take 5-20 μl of fresh or anticoagulated blood into a 1.5 ml centrifuge tube, and make up to 200 μl with Buffer DA.
c. Tissue culture cells: Collect approximately 10⁵-10⁶ suspension cells (total cell count should not exceed 5×10⁶) into a 1.5 ml centrifuge tube; for adherent cells, first digest with trypsin and resuspend by pipetting. Centrifuge at 12,000 rpm (~13,400×g) for 1 min, discard the supernatant, add 200 μl Buffer DA to the cell pellet, and vortex until completely resuspended.
d. Animal tissue: Weigh < 25 mg of animal tissue (spleen should be less than 10 mg), grind into fine powder with liquid nitrogen or cut into small pieces with a scalpel, and transfer to a 1.5 ml centrifuge tube pre-filled with 180 μl Buffer DA.
In addition to efficiently extracting DNA from blood, cells, and tissues, this kit can also extract high-quality DNA from bacteria.
e. Bacteria: Take 1-5 ml of bacterial culture, centrifuge at 12,000 rpm (~13,400×g) for 1 min, and aspirate the supernatant as much as possible.
1)For gram-positive bacteria with difficult cell wall lysis, add lysozyme solution for cell wall disruption: Add 110 μl buffer (20 mM Tris, pH 8.0; 2 mM Na₂-EDTA; 1.2% Triton) and 70 μl lysozyme solution (50 mg/ml, provided by the user), and incubate at 37°C for at least 30 min.
2)For relatively easily lysed bacteria, directly add 200 μl Buffer DA to the bacterial pellet and vortex until the bacteria are completely resuspended.
2. Optional step: If RNA removal is required (for cells, tissues, and bacteria), add 4 μl RNase A (100 mg/ml) solution (Catalog No.: R665518). Vortex for 15 sec and incubate at room temperature for 5 min.
3. Add 20 μl Proteinase K and vortex to mix thoroughly.
a. For blood, cell, or bacterial samples, simply add Proteinase K, mix well, and proceed to the next step.
b. For animal tissue samples, add Proteinase K and vortex to mix. Incubate at 56°C until the tissue is completely dissolved, then briefly centrifuge to collect the solution on the inner wall of the tube cap before proceeding to the next step.
Note: Lysis time varies for different tissues, usually taking 1-3 hours (mouse tail requires 6-8 hours of digestion, and overnight digestion is necessary if needed), which will not affect subsequent operations.
4. Add 200 μl Buffer DLB, invert thoroughly to mix, incubate at 70°C for 10 min, and briefly centrifuge to collect the solution on the inner wall of the tube cap.
Note: White precipitate may form when adding Buffer DLB, but it usually disappears during incubation at 70°C and will not affect subsequent experiments.
5. Add 200 μl absolute ethanol and mix (precipitate may appear at this point). Transfer the resulting solution and precipitate to the adsorption column Spin Columns GH4 (place the column in the collection tube), centrifuge at 12,000 rpm (~13,400×g) for 30 sec, discard the filtrate, and return the adsorption column to the collection tube.
6. Add 500 μl Buffer DW1 (ensure absolute ethanol is added before use) to the adsorption column, centrifuge at 12,000 rpm (~13,400×g) for 30 sec, discard the waste liquid, and return the adsorption column to the collection tube.
7. Add 500 μl Buffer DW2Plus (ensure absolute ethanol is added before use) to the adsorption column, centrifuge at 12,000 rpm (~13,400×g) for 30 sec, discard the waste liquid, and return the adsorption column to the collection tube.
8. Repeat step 7 once.
9. Centrifuge at 12,000 rpm for 3 min, and discard the waste liquid in the collection tube.
10. Transfer the adsorption column to a new 1.5 ml centrifuge tube, 悬空滴加 60-200 μl Buffer EB to the center of the adsorption membrane, let stand at room temperature for 1 min, and centrifuge at 12,000 rpm (~13,400×g) for 1 min to obtain the DNA solution.
Note: The volume of Buffer EB should not be less than 60 μl, as a smaller volume will affect recovery efficiency. If water is used as the eluent, ensure its pH is within the range of 7.0-8.5 (NaOH can be used to adjust the pH of water to this range). Store the DNA solution at -20°C.
Frequently Asked Questions and Solutions:
| Frequently Asked Questions | Cause | Solution |
| Column blockage | 1. Too much sample input | Please use samples within the compatible range. |
| 2. Inadequate Sample Lysis | Extend the 56°C water bath time and increase the number of inversion and mixing times. | |
| Low DNA Yield | 1. The sample has been repeatedly frozen and thawed | Avoid repeated freezing and thawing of samples. It is recommended to use fresh samples or samples that have been frozen frozen and thawed only once. |
| 2. Inadequate lysis of animal tissue | Cut tissue blocks into small pieces as much as possible or grind them with liquid nitrogen. During extraction, ensure that the samples are fully mixed with Buffer DA and Proteinase K in a timely manner, and the 56°C water bath lysis time can be appropriately extended. | |
| 3. Incomplete transfer of the lysis mixture into the adsorption column | After cell lysis, add absolute ethanol, flocculent precipitates will appear in the solution, which need to be transferred to the adsorption column together with the solution. | |
| 4. Issues with the eluent | Please use the Buffer EB provided with the kit for elution. If using ddH₂O or other eluents, ensure that the pH value of the eluent is between 7.0 - 8.0. Buffer EB can be preheated to 55°C in advance before elution, which is beneficial to improve the DNA yield. | |
| 5. Low elution efficiency | The eluent should be dropped in the center of the membrane; increase the elution volume or the number of elutions. | |
| 6. Failure to add absolute ethanol to Buffer DW1/DW2Plus | Add the specified volume of absolute ethanol to Buffer DW1 and Buffer DW2Plus as indicated on the bottle labels. | |
| Low DNA Purity | 1. Contamination with foreign proteins | Buffer DW1 was not used for rinsing, or the correct volume of absolute ethanol was not added to Buffer DW1. Please add the specified volume of absolute ethanol according to the bottle label before operation. |
| 2. Contamination with impurity ions | Buffer DW2Plus was not used for rinsing, or rinsed only once. Please rinse twice with Buffer DW2Plus according to the instruction manual. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0939452 |
分析证书 | M665559 |
| ZJ26F0333292 |
分析证书 | M665559 |
| ZJ25F0928165 |
分析证书 | M665559 |
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