本试剂盒采用环保脱蜡的方式去除石蜡，通过优化的缓冲液体系可高效释放组织切片中的DNA，同时克服了福尔马林交联所导致的抑制因素。提取得的DNA可适用于多种下游应用，如PCR、Real-time PCR、SNP基因分析、STR基因分析和药物基因组学研究等。
产品号
| 组件
| 物理外观
| 50T | 储存温度
|
M1522715A
| 环保脱蜡液
| 液体
| 33 mL
| RT.
|
M1522715B
| Buffer DF
| 液体
| 12 mL
| RT.
|
M1522715C
| Buffer DFB
| 液体
| 12 mL
| RT.
|
M1522715D
| Buffer RB
| 液体
| 20 mL
| RT.
|
M1522715E
| Buffer FPW
| 液体
| 38 mL | RT.
|
M1522715F
| Buffer EB
| 液体
| 15 mL | RT.
|
M1522715G
| 磁珠RS
| 液体
| 2.2 mL | RT.
|
M1522715H
| 蛋白酶K
| 液体
| 1.2 mL | 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. 实验需客户自备800 μL 80%乙醇；若残留RNA对后续实验有影响，需额外自备RNase A溶液 (100 mg/mL)、离心管、移液器吸头、手术刀、磁力架、旋转混匀仪等辅助工具。
2. 使用前请检查Buffer DF是否出现结晶或者沉淀，如有结晶或者沉淀，请将其置于37℃水浴中重新溶解后使用。
3. 所有试剂使用前请摇匀，避免试剂沉淀或磁珠聚集影响实验效果；试剂使用后请及时盖紧瓶盖，置于室温干燥处密封保存。
操作步骤
1. 样本处理
(1) 石蜡切片：取石蜡切片5-8张（5-10 μm厚，1×1 cm²大小），置于1.5 mL离心管中。
(2) 石蜡包埋样本：用手术刀刮取约10 mg的组织样本（尽量去除多余的石蜡），置于1.5 mL离心管中。
【注意】如果样品表面暴露于空气中，请将接触空气的2-3片弃掉不用。
2. 向离心管中加入600 μL环保脱蜡液，涡旋混匀，56℃孵育3 min；取出后剧烈涡旋15-30 sec，让石蜡充分溶解。
3. 14,000 rpm离心2 min，小心吸弃上清液（避免吸弃管底沉淀）；瞬时离心后，离心管底部剩余的脱蜡液可用200 μL枪头吸净。
【注意】若脱蜡不彻底，可重复步骤2-3一次。
4. 向离心管中加入200 μL Buffer DF和20 μL蛋白酶K，涡旋振荡5-10 sec后，瞬时离心，确保试剂与样本充分接触，避免试剂残留于管盖或管壁。
5. 56℃孵育1 h，直至样品完全裂解；【注意】该步骤可能会出现样本无法完全裂解的情况，直接进入下一步骤即可。
6. 70℃孵育过夜。
7. 瞬时离心后，使用200 μL枪头沿管壁小心吸取下层200 μL孵育产物，转移至新的1.5 mL离心管中，避免吸入杂质。
8. 若残留RNA对后续实验有影响，可向孵育产物中加入5 μL RNase A溶液 (100 mg/mL)，振荡混匀，室温放置5-10 min，去除RNA杂质。
9. 向装有孵育产物的离心管中，加入200 μL Buffer DFB、200 μL Buffer RB和40 μL磁珠RS（磁珠悬液使用前应充分振荡混匀）；涡旋混匀20 sec，置于旋转混匀仪上孵育10 min，或静置10-15 min（静置期间每隔2-3 min涡旋混匀20 sec），确保DNA与磁珠充分结合。
10. 瞬时离心后，将离心管置于磁力架上静置，待磁珠完全吸附（溶液澄清），小心吸弃上清液，避免吸弃磁珠。
11. 向离心管中加入700 μL Buffer FPW，振荡混匀1 min，确保磁珠完全分散，无结块；瞬时离心后，将离心管置于磁力架上静置，待磁珠完全吸附，小心吸弃上清，避免吸弃磁珠。
12. 向离心管中加入700 μL 80%乙醇（客户自备），振荡混匀1 min，确保磁珠完全分散；瞬时离心后，将离心管置于磁力架上静置，待磁珠完全吸附，小心吸弃上清，避免吸弃磁珠。
13. 重复步骤12。
14. 尽量吸净离心管内残留的液体，将离心管置于磁力架上晾干5-10 min，或60℃加热晾干，直至乙醇完全挥发。【注意】晾干至磁珠表面刚出现龟裂即可，过度干燥不利于核酸洗脱。
15. 将离心管从磁力架上取下，加入50-100 μL Buffer EB，振荡混匀；如有磁珠结块，可用移液器轻轻吹打分散。瞬时离心后，70℃孵育3 min，期间可振荡混匀1-2次，以提高DNA洗脱效率。
16. 瞬时离心后，将离心管放置于磁力架上静置，待磁珠完全吸附，小心将洗脱液转移至新的离心管中，避免吸到磁珠，获得纯化的基因组DNA。
17. 可将洗脱液置于-20℃短期保存或-80℃长期保存，避免反复冻融
This kit uses an environmentally friendly deparaffinization method to remove paraffin. The optimized buffer system efficiently releases DNA from tissue sections while overcoming inhibitory factors caused by formalin crosslinking. The extracted DNA is suitable for various downstream applications, such as PCR, real-time PCR, SNP genotyping, STR analysis, and pharmacogenomics research.
| Product No. | Component | Appearance | 50T | Storage |
|---|
| M1522715A | Eco-Friendly Deparaffinization Solution | Liquid | 33 mL | RT | | M1522715B | Buffer DF | Liquid | 12 mL | RT | | M1522715C | Buffer DFB | Liquid | 12 mL | RT | | M1522715D | Buffer RB | Liquid | 20 mL | RT | | M1522715E | Buffer FPW | Liquid | 38 mL | RT | | M1522715F | Buffer EB | Liquid | 15 mL | RT | | M1522715G | Magnetic Beads RS | Liquid | 2.2 mL | RT | | M1522715H | Proteinase K | Liquid | 1.2 mL | RT |
|
All components of this kit can be stored under dry conditions at room temperature for 15 months.
Precautions
The user needs to prepare 800 μL of 80% ethanol. If residual RNA affects downstream applications, additional self-supplied items include RNase A solution (100 mg/mL), centrifuge tubes, pipette tips, a scalpel, a magnetic stand, a rotator mixer, and other auxiliary tools.
Before use, check Buffer DF for crystallization or precipitation. If present, redissolve by placing in a 37°C water bath before use.
Shake all reagents before use to avoid precipitation or magnetic bead aggregation that may affect results. After use, cap the bottles tightly and store sealed in a dry place at room temperature.
Procedure
1. Sample Processing
(1) Paraffin sections: Take 5–8 paraffin sections (5–10 μm thick, 1×1 cm² each) and place into a 1.5 mL centrifuge tube.
(2) Paraffin-embedded samples: Use a scalpel to scrape approximately 10 mg of tissue (remove excess paraffin as much as possible) and place into a 1.5 mL centrifuge tube.
【Note】If the sample surface has been exposed to air, discard the 2–3 sections that came into contact with air.
2. Add 600 μL of Eco-Friendly Deparaffinization Solution to the tube, vortex to mix, and incubate at 56°C for 3 min. Remove and vortex vigorously for 15–30 sec to fully dissolve the paraffin.
3. Centrifuge at 14,000 rpm for 2 min. Carefully aspirate and discard the supernatant (avoid disturbing the pellet). After a quick spin, remove any remaining deparaffinization solution at the bottom of the tube using a 200 μL pipette tip.
【Note】If deparaffinization is incomplete, repeat steps 2–3 once.
4. Add 200 μL of Buffer DF and 20 μL of Proteinase K to the tube. Vortex for 5–10 sec, then briefly centrifuge to ensure reagents fully contact the sample and no residue remains on the cap or tube wall.
5. Incubate at 56°C for 1 h until the sample is completely lysed. 【Note】If the sample does not fully lyse at this step, proceed directly to the next step.
6. Incubate at 70°C overnight.
7. Briefly centrifuge, then carefully transfer 200 μL of the lower layer of the incubation product using a 200 μL pipette tip along the tube wall to a new 1.5 mL centrifuge tube, avoiding any impurities.
8. If residual RNA affects downstream applications, add 5 μL of RNase A solution (100 mg/mL) to the incubation product, mix by vortexing, and let stand at room temperature for 5–10 min to remove RNA impurities.
9. Add 200 μL of Buffer DFB, 200 μL of Buffer RB, and 40 μL of Magnetic Beads RS (shake the bead suspension thoroughly before use) to the tube containing the incubation product. Vortex for 20 sec, then incubate on a rotator mixer for 10 min, or let stand for 10–15 min (vortexing for 20 sec every 2–3 min during standing) to ensure full binding of DNA to the magnetic beads.
10. Briefly centrifuge, then place the tube on a magnetic stand and wait until the beads are completely attracted (solution becomes clear). Carefully aspirate and discard the supernatant without disturbing the beads.
11. Add 700 μL of Buffer FPW to the tube, vortex for 1 min to fully disperse the beads without clumping. Briefly centrifuge, place the tube on the magnetic stand, wait for beads to be fully attracted, then carefully aspirate and discard the supernatant without disturbing the beads.
12. Add 700 μL of 80% ethanol (user-supplied) to the tube, vortex for 1 min to fully disperse the beads. Briefly centrifuge, place the tube on the magnetic stand, wait for beads to be fully attracted, then carefully aspirate and discard the supernatant without disturbing the beads.
13. Repeat step 12.
14. Remove as much residual liquid as possible from the tube. Place the tube on the magnetic stand and air-dry for 5–10 min, or heat-dry at 60°C until the ethanol has completely evaporated. 【Note】Dry until the bead surface just begins to crack; over-drying is unfavorable for nucleic acid elution.
15. Remove the tube from the magnetic stand. Add 50–100 μL of Buffer EB and vortex to mix. If beads have clumped, gently pipette to disperse. Briefly centrifuge, then incubate at 70°C for 3 min, mixing by vortexing 1–2 times during incubation to improve DNA elution efficiency.
16. Briefly centrifuge, place the tube on the magnetic stand, wait for beads to be fully attracted, then carefully transfer the eluate to a new centrifuge tube without carrying over any beads. The purified genomic DNA is obtained.
17. The eluate can be stored short‑term at –20°C or long‑term at –80°C. Avoid repeated freeze‑thaw cycles.
ZJ26F0636708
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