本试剂盒采用可特异性结合核酸的离心吸附柱和独特的缓冲液系统，样品经裂解后，DNA在高盐条件下与硅胶膜结合，在低盐、高pH值条件下DNA从硅胶膜上洗脱下来。本试剂盒适用于从少量血液、干血斑、血清/血浆、微量组织、漱口水、毛发和微切割组织等微量样品中提取基因组DNA，所得基因组DNA可直接用作PCR模板、酶切和杂交等分子生物学实验。
产品号
| 组件
| 物理外观
| 50T | 储存温度
|
M1522624A
| Buffer UA
| 液体
| 15 mL
| RT.
|
M1522624B
| Buffer BDL
| 液体
| 15 mL
| RT.
|
M1522624C
| Buffer PW1
| 液体
| 17 mL
| RT.
|
M1522624D
| Buffer PW2
| 液体
| 15 mL
| RT.
|
M1522624E
| Buffer EB
| 液体
| 15 mL
| RT.
|
M1522624F
| 蛋白酶K
| 液体
| 1.2 mL
| RT.
|
M1522624G
| 吸附柱GHT (含2mL收集管)
| —
| 1 EA×50 | RT.
|
M1522624H
| 核酸增强剂
| 液体
| 280 μL | -20℃. |
该试剂盒置于室温干燥条件下，可保存15个月。
核酸增强剂冰袋运输，4℃可存放1个月，长期保存置于-20℃保存。
注意事项
1. 实验需自备无水乙醇、1M DTT（用于提取毛囊毛发）、RNase A (10 mg/mL)、离心管、无菌管、打孔器等辅助工具。
2. 所有的样品使用前请平衡到室温；如果室温超过25℃，需要先将无水乙醇置于冰上预冷，避免影响实验效果。
3. 为确保从微量样本中得到更多的DNA，本试剂盒配备了核酸增强剂；由于核酸增强剂本身是小核酸，因此得到的基因组测定OD<sub>260</sub>值会比真实值偏大，建议将得到的基因组直接用PCR进行检测。
4. 使用前请先在Buffer PW1和Buffer PW2中加入无水乙醇，Buffer UA在低温下会有沉淀析出，可在37℃水浴中重新溶解，摇匀后使用。
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。
本试剂盒针对不同微量样本设计专属提取流程，按样品类型选择对应步骤操作，所有样本完成对应步骤后，均接步骤8继续操作。
1. 微量血液样品
(1) 取1–100 µL血液到1.5 mL的离心管中，若血液体积不足100 μL，用Buffer UA补足至100 μL。
(2) 如果需要去除RNA，可加入5 μL RNase A (10 mg/mL) 溶液（需自备），振荡混匀，室温放置5 min，去除RNA杂质。
(3) 加入10 µL蛋白酶K，涡旋振荡混匀，确保酶与样品充分接触。
(4) 加入100 µL Buffer BDL，充分颠倒混匀，56℃孵育10 min，期间应混匀样品2-3次；孵育结束后短暂离心，收集管盖内壁上的溶液，避免试剂残留。
【注意】如果血液样品体积<10 µL，请在100 µL Buffer BDL中加入5 µL 核酸增强剂。加入Buffer BDL时可能会产生白色沉淀，一般56℃孵育时会消失，不会影响后续实验；如溶液未变清亮，说明细胞裂解不彻底，可能导致提取的DNA产量和纯度降低。
(5) 加入50 µL无水乙醇，轻轻颠倒混匀，室温放置3 min；简短离心，去除管盖内壁液滴。
(6) 接步骤8继续进行实验。
2. 干血斑
(1) 用打孔器打孔或剪裁的方法，取三片3×3 mm的干血斑样品，加入到1.5 mL的离心管中。
(2) 加入180 µL Buffer UA，确保样品完全浸泡在缓冲液中。
(3) 加入20 µL蛋白酶K，涡旋振荡混匀；56℃孵育60 min，期间应每10 min颠倒混匀一次，促进样品裂解。
(4) 加入200 µL Buffer BDL，涡旋振荡混匀，70℃孵育10 min，期间应每3 min涡旋混匀一次；孵育结束后短暂离心，收集管盖内壁上的溶液。
【注意】加入Buffer BDL时可能会产生白色沉淀，一般70℃孵育时会消失，不会影响后续实验；如溶液未变清亮，说明细胞裂解不彻底，可能导致提取的DNA量少和不纯。
(5) 加入200 µL无水乙醇，轻轻颠倒混匀样品，室温放置5 min；简短离心，去除管盖内壁液滴。
(6) 接步骤8继续进行实验。
3. 微量组织
(1) 称取不超过10 mg的动物组织，转入预先装有180 μL Buffer UA的1.5 mL离心管中。
(2) 加入20 µL蛋白酶K，涡旋振荡混匀；56℃孵育30 min至60 min，期间应每15 min颠倒混匀一次，确保组织充分裂解。
(3) 加入200 µL Buffer BDL和5 µL 核酸增强剂，涡旋振荡混匀，70℃孵育10 min，期间应每3 min涡旋混匀一次；溶液变清亮后短暂离心，收集管盖内壁上的溶液。
(4) 加入200 µL无水乙醇，轻轻颠倒混匀样品，室温放置5 min；简短离心，去除管盖内壁液滴。
(5) 接步骤8继续进行实验。
4. 微切割样品（包括福尔马林固定的微切割样品）
(1) 加入15 µL Buffer UA到0.2 mL离心管中，放入微切割样品。
(2) 加入10 µL蛋白酶K，涡旋振荡混匀，确保样品与酶充分接触。
(3) 56℃孵育 3h（福尔马林固定样品需要孵育16 h），直至样品裂解完全，期间应不时混匀样品。
(4) 加入25 µL Buffer UA并混匀，再加入50 µL Buffer BDL和5 µL 核酸增强剂，涡旋振荡混匀；短暂离心，收集管盖内壁上的溶液。
(5) 加入50 µL无水乙醇，轻轻颠倒混匀样品，室温放置5 min。
(6) 接步骤8继续进行实验。
5. 法医材料（毛发、沾染体液材料、指甲）
(1) 样本处理
- 毛发：从毛发根部毛囊处取1 cm长度毛发放入1.5 mL离心管，加入250 µL Buffer UA、2 0µL蛋白酶K和20 µL 1M DTT溶液，涡旋振荡混匀，接步骤 (2)。
- 沾染体液材料：取约0.5 cm³沾染了血液、唾液或精液的材料并剪成小块，放入1.5 mL离心管，加入300 µL Buffer UA和20 µL蛋白酶K，涡旋振荡混匀，接步骤 (2)。【注意】如果样本是精液，还需要额外加入20 µL 1M DTT溶液。
- 指甲：将指甲剪成小块放入1.5 mL离心管，加入300 µL Buffer UA、20 µL蛋白酶K和20 µL 1M DTT溶液，涡旋振荡混匀，接步骤 (2)。
(2) 56℃孵育至样本完全裂解，每10 min应颠倒混匀一次。【注意】一般毛发60 min即可完成裂解，指甲等难裂解物建议过夜裂解。
(3) 加入300 µL Buffer BDL和5 µL 核酸增强剂，涡旋振荡混匀；56℃水浴10 min，期间每3 min涡旋混匀一次；短暂离心，收集管盖内壁上的溶液。
(4) 加入300 µL无水乙醇，轻轻颠倒混匀样品，室温放置3 min。
(5) 接步骤8继续进行实验。
6. 从血清/血浆中提取循环核酸/游离核酸
(1) 取100–200 µL血清/血浆到1.5 mL的离心管中，若体积不足100 μL，用Buffer UA补足。
(2) 加入20 µL蛋白酶K，涡旋振荡混匀。
(3) 加入200 µL Buffer BDL，充分颠倒混匀，56℃孵育10 min，期间应混匀样品2-3次；短暂离心，收集管盖内壁上的溶液。【注意】如果血清/血浆样品体积<50 µL，请在200 µL Buffer BDL中加入5 µL 核酸增强剂。加入Buffer BDL时可能会产生白色沉淀，一般56℃孵育时会消失，不会影响后续实验。
(4) 加入200 µL无水乙醇，轻轻颠倒混匀，室温放置5 min。
(5) 接步骤8继续进行实验。
7. 从漱口水中提取基因组DNA
(1) 在50 mL无菌管中添加10-20 mL漱口水样品，800 rpm离心5 min，小心倒掉上清液，保留底部沉淀。
(2) 向沉淀中添加200 µL Buffer UA，充分重悬沉淀；将全部悬液转移至1.5 mL离心管中。
(3) 加入20 µL蛋白酶K，涡旋混匀；56℃孵育60 min，期间每15 min涡旋混匀一次，促进沉淀裂解。
(4) 加入200 µL Buffer BDL和5 µL 核酸增强剂，充分颠倒混匀，70℃孵育10 min，期间应每3 min涡旋混匀一次；待溶液变清亮后短暂离心，收集管盖内壁上的溶液。【注意】加入Buffer BDL时可能会产生白色沉淀，一般70℃孵育时会消失，不会影响后续实验；如溶液未变清亮，说明细胞裂解不彻底，可能导致提取的DNA得率和纯度低。
(5) 加入200 µL无水乙醇，充分颠倒混匀样品，简短离心，去除管盖内壁液滴。
(6) 接步骤8继续进行实验。
8. 将上述各样本对应步骤所得的溶液转入吸附柱GHT（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。
9. 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。
10. 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。
11. 重复步骤10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。
12. 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）。
13. 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。向吸附膜中间部位缓慢悬空滴加20 μL Buffer EB，室温放置1 min；12,000 rpm (13,680×g) 离心30 sec，收集DNA溶液，可立即使用或-20℃长期保存。【注意】若要提高DNA的产量，可将第一次洗脱液重新加入吸附柱进行洗脱，或将Buffer EB置于65℃预热后使用。
This kit uses a spin column that specifically binds nucleic acids and a unique buffer system. After sample lysis, DNA binds to the silica membrane under high‑salt conditions and is eluted from the membrane under low‑salt, high‑pH conditions. This kit is suitable for extracting genomic DNA from small amounts of samples such as low‑volume blood, dried blood spots, serum/plasma, micro‑tissues, mouthwash, hair, and microdissected tissues. The obtained genomic DNA can be directly used in molecular biology experiments such as PCR templates, restriction enzyme digestion, and hybridization.
| Product No. | Component | Appearance | 50T | Storage |
|---|
| M1522624A | Buffer UA | Liquid | 15 mL | RT | | M1522624B | Buffer BDL | Liquid | 15 mL | RT | | M1522624C | Buffer PW1 | Liquid | 17 mL | RT | | M1522624D | Buffer PW2 | Liquid | 15 mL | RT | | M1522624E | Buffer EB | Liquid | 15 mL | RT | | M1522624F | Proteinase K | Liquid | 1.2 mL | RT | | M1522624G | GHT Spin Column (with 2 mL collection tube) | — | 1 EA × 50 | RT | | M1522624H | Trace RNA Enhancer | Liquid | 280 μL | –20°C |
|
The kit can be stored under dry conditions at room temperature for 15 months.
The Trace RNA Enhancer is shipped with ice packs; it can be stored at 4°C for 1 month and at –20°C for long‑term storage.
Precautions
The user needs to supply absolute ethanol, 1 M DTT (for hair follicle extraction), RNase A (10 mg/mL), centrifuge tubes, sterile tubes, a hole punch, and other auxiliary tools.
Bring all samples to room temperature before use. If the room temperature exceeds 25°C, pre‑cool the absolute ethanol on ice to avoid affecting the results.
To maximize DNA yield from trace samples, this kit includes a Trace RNA Enhancer. Because the Enhancer itself is a small nucleic acid, the measured OD260 value of the genomic DNA will be higher than the actual value. It is recommended to directly test the obtained genomic DNA by PCR.
Before use, add absolute ethanol to Buffer PW1 and Buffer PW2. Buffer UA may precipitate at low temperatures; redissolve in a 37°C water bath and shake well before use.
Procedure
Before use, add 22 mL of absolute ethanol to Buffer PW1 and 60 mL of absolute ethanol to Buffer PW2.
This kit provides dedicated extraction protocols for different trace sample types. Follow the respective steps according to the sample type. After completing the corresponding steps, all samples proceed to step 8.
1. Low‑volume blood samples
(1) Transfer 1–100 µL of blood to a 1.5 mL centrifuge tube. If the blood volume is less than 100 μL, bring the volume to 100 μL with Buffer UA.
(2) If RNA removal is required, add 5 μL of RNase A (10 mg/mL) solution (user‑supplied), vortex to mix, and let stand at room temperature for 5 min.
(3) Add 10 µL of Proteinase K and vortex to ensure thorough contact between the enzyme and the sample.
(4) Add 100 µL of Buffer BDL, mix thoroughly by inversion, and incubate at 56°C for 10 min, mixing the sample 2–3 times during incubation. After incubation, briefly centrifuge to collect the solution from the cap wall, avoiding reagent residue.
【Note】 If the blood sample volume is <10 µL, add 5 µL of Trace RNA Enhancer to the 100 µL of Buffer BDL. White precipitate may form upon addition of Buffer BDL, but usually disappears during incubation at 56°C and does not affect subsequent steps. If the solution does not become clear, cell lysis is incomplete, which may lead to reduced DNA yield and purity.
(5) Add 50 µL of absolute ethanol, mix gently by inversion, and let stand at room temperature for 3 min. Briefly centrifuge to remove drops from the cap wall.
(6) Proceed to step 8.
2. Dried blood spots
(1) Using a hole punch or scissors, take three 3×3 mm pieces of dried blood spot sample and place into a 1.5 mL centrifuge tube.
(2) Add 180 µL of Buffer UA to completely immerse the sample.
(3) Add 20 µL of Proteinase K, vortex to mix, and incubate at 56°C for 60 min, inverting to mix every 10 min to promote lysis.
(4) Add 200 µL of Buffer BDL, vortex to mix, and incubate at 70°C for 10 min, vortexing every 3 min. After incubation, briefly centrifuge to collect the solution from the cap wall.
【Note】 White precipitate may form upon addition of Buffer BDL, but usually disappears during incubation at 70°C. If the solution does not become clear, lysis is incomplete, which may lead to low DNA yield and purity.
(5) Add 200 µL of absolute ethanol, mix gently by inversion, and let stand at room temperature for 5 min. Briefly centrifuge to remove drops from the cap wall.
(6) Proceed to step 8.
3. Micro‑tissue samples
(1) Weigh no more than 10 mg of animal tissue and transfer to a 1.5 mL centrifuge tube pre‑filled with 180 μL of Buffer UA.
(2) Add 20 µL of Proteinase K, vortex to mix, and incubate at 56°C for 30–60 min, inverting to mix every 15 min to ensure complete tissue lysis.
(3) Add 200 µL of Buffer BDL and 5 µL of Trace RNA Enhancer, vortex to mix, and incubate at 70°C for 10 min, vortexing every 3 min. After the solution becomes clear, briefly centrifuge to collect the solution from the cap wall.
(4) Add 200 µL of absolute ethanol, mix gently by inversion, and let stand at room temperature for 5 min. Briefly centrifuge to remove drops from the cap wall.
(5) Proceed to step 8.
4. Microdissected samples (including formalin‑fixed microdissected samples)
(1) Add 15 µL of Buffer UA to a 0.2 mL centrifuge tube and place the microdissected sample into the tube.
(2) Add 10 µL of Proteinase K and vortex to ensure thorough contact.
(3) Incubate at 56°C for 3 h (formalin‑fixed samples require 16 h) until the sample is completely lysed, mixing occasionally.
(4) Add 25 µL of Buffer UA and mix, then add 50 µL of Buffer BDL and 5 µL of Trace RNA Enhancer, vortex to mix. Briefly centrifuge to collect the solution from the cap wall.
(5) Add 50 µL of absolute ethanol, mix gently by inversion, and let stand at room temperature for 5 min.
(6) Proceed to step 8.
5. Forensic materials (hair, body fluid‑stained materials, nails)
(1) Sample processing
Hair: Take 1 cm of hair from the root (including the follicle) and place into a 1.5 mL centrifuge tube. Add 250 µL of Buffer UA, 20 µL of Proteinase K, and 20 µL of 1 M DTT solution. Vortex to mix. Proceed to step (2).
Body fluid‑stained material: Take approximately 0.5 cm³ of material stained with blood, saliva, or semen, cut into small pieces, and place into a 1.5 mL centrifuge tube. Add 300 µL of Buffer UA and 20 µL of Proteinase K. Vortex to mix. Proceed to step (2). 【Note】For semen samples, additionally add 20 µL of 1 M DTT solution.
Nails: Cut nails into small pieces and place into a 1.5 mL centrifuge tube. Add 300 µL of Buffer UA, 20 µL of Proteinase K, and 20 µL of 1 M DTT solution. Vortex to mix. Proceed to step (2).
(2) Incubate at 56°C until the sample is completely lysed, inverting to mix every 10 min. 【Note】Hair usually lyses in 60 min; tough materials such as nails may require overnight lysis.
(3) Add 300 µL of Buffer BDL and 5 µL of Trace RNA Enhancer, vortex to mix, and incubate at 56°C for 10 min, vortexing every 3 min. Briefly centrifuge to collect the solution from the cap wall.
(4) Add 300 µL of absolute ethanol, mix gently by inversion, and let stand at room temperature for 3 min.
(5) Proceed to step 8.
6. Circulating cell‑free nucleic acid extraction from serum/plasma
(1) Transfer 100–200 µL of serum/plasma to a 1.5 mL centrifuge tube. If the volume is less than 100 μL, bring to volume with Buffer UA.
(2) Add 20 µL of Proteinase K and vortex to mix.
(3) Add 200 µL of Buffer BDL, mix thoroughly by inversion, and incubate at 56°C for 10 min, mixing the sample 2–3 times during incubation. Briefly centrifuge to collect the solution from the cap wall. 【Note】If the serum/plasma volume is <50 µL, add 5 µL of Trace RNA Enhancer to the 200 µL of Buffer BDL. White precipitate may form upon addition of Buffer BDL, but usually disappears during incubation at 56°C.
(4) Add 200 µL of absolute ethanol, mix gently by inversion, and let stand at room temperature for 5 min.
(5) Proceed to step 8.
7. Genomic DNA extraction from mouthwash
(1) Add 10–20 mL of mouthwash sample to a 50 mL sterile tube. Centrifuge at 800 rpm for 5 min. Carefully pour off the supernatant, retaining the pellet at the bottom.
(2) Add 200 µL of Buffer UA to the pellet and resuspend thoroughly. Transfer the entire suspension to a 1.5 mL centrifuge tube.
(3) Add 20 µL of Proteinase K and vortex to mix. Incubate at 56°C for 60 min, vortexing every 15 min to promote lysis of the pellet.
(4) Add 200 µL of Buffer BDL and 5 µL of Trace RNA Enhancer, mix thoroughly by inversion, and incubate at 70°C for 10 min, vortexing every 3 min. After the solution becomes clear, briefly centrifuge to collect the solution from the cap wall. 【Note】White precipitate may form upon addition of Buffer BDL, but usually disappears during incubation at 70°C. If the solution does not become clear, lysis is incomplete, which may lead to low yield and purity.
(5) Add 200 µL of absolute ethanol, mix thoroughly by inversion, and briefly centrifuge to remove drops from the cap wall.
(6) Proceed to step 8.
8. Transfer the solution obtained from the respective sample steps above into a GHT 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.
9. 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.
10. 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.
11. Repeat step 10 to further remove residual salts, proteins, and other impurities on the spin column, thereby increasing DNA purity.
12. 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).
13. 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 20 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. The DNA can be used immediately or stored at –20°C for long‑term preservation.
【Note】 To increase DNA yield, reload the first eluate onto the spin column and repeat the elution, or pre‑heat Buffer EB to 65°C before use.
H2614321
危险品化学品经营许可证（带存储）