BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
2-8°C储存,避光,室温,-20°C储存,避免反复冻融。常规运输,低温运输,超低温运输 。请查阅批次 COA 获取详细规格。
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
用分子生物学技术研究复杂的基因组，首先要求制备纯净的高分子质量 DNA，一般分为两个步骤，先裂解细胞、溶解 DNA，接着采用酶学或化学方法去除蛋白、RNA 以及其他大分子。
本品采用经典的蛋白酶 K 处理法，可以抽提到 100～150kb 以上的基因组 DNA，提取的基因组 DNA 可用于 Southern 杂交、基因组 DNA 的 PCR 扩增及基因组 DNA 文库的构建等。该试剂盒的提取效率大致为：从 1g 组织可以提取到约 150～200μg 100kb 以上的基因组DNA，从 106～107Hela 细胞可以提取到约 5～50μg 100kb 以上的基因组DNA。
该试剂仅用于科研领域，不适用于临床诊断或其他用途。
组分表
|
自备材料
1、 组织或培养细胞
2、 PBS、无水乙醇、70%乙醇、25:24:1 酚/氯仿/异戊醇
3、 离心机、研钵、剪刀、恒温箱、EP 管
操作步骤(仅供参考)
1、准备样品：
①组织样本：切下组织并立即剪成小块，置于液氮中冻结，将100mg冻结的组织用预冷的研钵和研杵研碎或用锤子将其捣成碎末；
②培养细胞：收集贴壁生长或悬浮生长细胞培养液，贴壁生长细胞需先用胰蛋白酶消化液消化，再用PBS清洗1次。4℃离心机，1500rpm离心1～2min，弃上清，用1～2ml预冷的PBS再次悬浮离心，如此2洗涤次。
2、每1ml样品裂解液加入5μl的蛋白酶K溶液，混匀。每50mg组织或5×10⁷细胞用0.45~0.55ml样品裂解液悬浮，悬浮应彻底，不应留下结块。
3、充分混匀，50℃振荡下温育12～18h或过夜。
4、加入等体积25:24:1酚/氯仿/异戊醇，轻轻混合，尽可能减少对DNA的剪切。
5、吸出酚相及中间相(可以吸除少量靠近中间相的水相液体)，剩余的水相用等体积酚/氯仿/异戊醇再抽提1次；重复该步骤1次。
6、吸取上层液(水溶液)250～300μl至新EP管中，加入等体积无水乙醇和1/4体积的乙酸铵溶液，颠倒混匀数次。4℃ 10000rpm离心1min，弃上清。
7、向沉淀中加入70%乙醇，4℃ 10000rpm离心1min，弃上清。重复该步骤一次。
8、尽量吸除残余的乙醇，空气干燥，等到不到明显液体时，立即加入50～100μl 无核酸酶水溶解DNA，4℃或-20℃保存。注意：不可过分干燥基因组DNA沉淀，否则会极难溶解；如果发现DNA沉淀难以溶解，可以在4℃用摇床缓慢摇动过夜，以溶解DNA沉淀。
9、A260/A280处检测DNA纯度，高纯度的DNA一般 A₂₆₀/A₂₈₀>1.8 。
注意事项
1、如果打算提取大片段的基因组DNA，应尽量避免基因组DNA的物理性剪切。例如避免剧烈振荡含有基因组DNA的样品，可以用剪掉枪头尖的枪头吸取基因组DNA的样品。
2、准备细胞样品时，如果细胞量过少(＜3×10⁷)，应0.3ml含蛋白酶K的样品裂解液。
3、为避免高分子质量DNA被剪切，可以不采用乙醇沉淀DNA，可用透析袋替代乙醇：在100倍体积的TE Buffer中至少透析2次，所用取全部时间应＞24h。
4、不可过分干燥基因组DNA沉淀，否则会极难溶解。
5、为了您的安全和健康，请穿实验服并戴一次性手套操作。
6、试剂开封后请尽快使用，以防影响后续实验效果。
To study complex genomes using molecular biological techniques, high‑purity high‑molecular‑weight (HMW) DNA must first be prepared. The procedure generally consists of two steps: cell lysis and DNA solubilization, followed by enzymatic or chemical removal of proteins, RNA and other macromolecules.
This kit adopts the classic Proteinase K digestion method to isolate genomic DNA larger than 100‑150 kb. Purified genomic DNA is suitable for Southern blotting, genomic DNA PCR amplification, and genomic DNA library construction. Typical extraction yields are as follows: approximately 150‑200 μg of >100 kb genomic DNA from 1 g tissue; approximately 5‑50 μg of >100 kb genomic DNA from 10⁶‑10⁷ HeLa cells.
For Research Use Only. Not intended for clinical diagnosis or other applications.
Component
| Storage Unit No. | Storage Unit Name | Component No. | Component Name | Appearance | 50T | Storage |
| M1527094NT | Mammalian Genomic DNA Extraction Kit (Proteinase K Method)(RT Components) | M1527094A | Ammonium Acetate Solution | Liquid | 10 mL | Room temperature. |
| M1527094B | Nuclease-Free Water | Liquid | 10 mL | Room temperature. | ||
| M1527094WT | Mammalian Genomic DNA Extraction Kit (Proteinase K Method)(2-8℃ Components) | M1527094C | Lysis Buffer | Liquid | 50 mL | 2-8℃. |
| M1527094DT | Mammalian Genomic DNA Extraction Kit (Proteinase K Method)(-20℃ Components) | M1527094D | Proteinase K Solution | Liquid | 500 μL | -20℃.Store in the dark. |
Note: Each component is individually packaged according to its storage temperature (Room temperature/2‑8 °C / -20 °C) upon shipment, with differentiated temperature‑controlled transportation to guarantee product performance. Orders containing materials requiring multiple temperature zones may be delivered in separate batches; please refer to the actual received goods. Upon receipt, store components separately at the temperatures specified in the component list. The shelf‑life is 12 months from the date of manufacture.
Reagents, consumables and Equipments not provided
1. Tissue or cultured cells
2. PBS, absolute ethanol, 70% ethanol, phenol‑chloroform‑isoamyl alcohol (25:24:1)
3. Centrifuge, mortar, scissors, incubator, EP tubes
Protocol (For Reference Only)
1. Sample preparation
① Tissue samples: Excise tissue and immediately cut into small pieces, freeze in liquid nitrogen. Grind 100 mg frozen tissue in pre‑chilled mortar and pestle, or crush into fine powder with a hammer.
② Cultured cells: Collect medium from adherent or suspension cells. Adherent cells shall be digested with trypsin solution prior to collection, then washed once with PBS. Centrifuge at 1500 rpm for 1‑2 min at 4 °C, discard supernatant. Resuspend pellet in 1‑2 mL pre‑chilled PBS and centrifuge; repeat this washing step twice.
2. Add 5 μL Proteinase K Solution per 1 mL Sample Lysis Buffer and mix well. Resuspend 50 mg tissue or 5×10⁷ cells in 0.45‑0.55 mL Sample Lysis Buffer. Ensure complete resuspension with no visible clumps.
3. Mix thoroughly and incubate at 50 °C with shaking for 12‑18 h or overnight.
4. Add an equal volume of phenol‑chloroform‑isoamyl alcohol (25:24:1). Mix gently to minimize DNA shearing.
5. Aspirate the phenol phase and interphase (a small volume of aqueous phase adjacent to the interphase may be removed). Re‑extract the remaining aqueous phase with an equal volume of phenol‑chloroform‑isoamyl alcohol; repeat once.
6. Transfer 250‑300 μL of the upper aqueous phase to a new EP tube. Add an equal volume of absolute ethanol and 1/4 volume of Ammonium Acetate Solution. Mix by inverting the tube several times. Centrifuge at 10 000 rpm for 1 min at 4 °C and discard supernatant.
7. Add 70% ethanol to the pellet. Centrifuge at 10 000 rpm for 1 min at 4 °C and discard supernatant. Repeat once.
8. Remove residual ethanol as completely as possible and air‑dry the pellet. Before the pellet becomes visibly dry, immediately add 50‑100 μL Nuclease‑Free Water to dissolve DNA. Store at 4 °C or −20 °C.
Note: Do not over‑dry the genomic DNA pellet, as it will become extremely difficult to dissolve. If the pellet is hard to resuspend, incubate overnight with gentle shaking at 4 °C.
9. Determine DNA purity by measuring A₂₆₀/A₂₈₀ ratio. High‑quality genomic DNA typically shows A₂₆₀/A₂₈₀ ＞ 1.8.
Precautions
1. When isolating large‑fragment genomic DNA, avoid physical shearing. Do not vortex vigorously genomic‑DNA‑containing samples. Use pipette tips with tips cut off for pipetting genomic DNA solutions.
2. For cell samples with low cell count (＜ 3×10⁷ cells), use 0.3 mL Sample Lysis Buffer pre‑mixed with Proteinase K.
3. To avoid shearing high‑molecular‑weight DNA, ethanol precipitation may be omitted. Instead, dialyze the sample in dialysis tubing against at least 100‑fold volume of TE Buffer for ≥ 2 times with total dialysis time ＞ 24 h.
4. Do not over‑dry the genomic DNA pellet; excessive drying will render it very difficult to dissolve.
5. For personal safety, wear lab coat and disposable gloves during operation.
6. Use reagents promptly after opening to avoid compromising experimental performance.
| 储运单元货号 | 储运单元名称 | 组分货号 | 组分名称 | 物理外观 | 50T | 储存 |
| M1527094NT | 哺乳动物基因组DNA抽提试剂盒 (蛋白酶K法) (常温组分) | M1527094A | 乙酸铵溶液 | 液体 | 10 mL | 室温 |
| M1527094B | 无核酸酶水 | 液体 | 10 mL | 室温 | ||
| M1527094WT | 哺乳动物基因组DNA抽提试剂盒 (蛋白酶K法) (2-8℃组分) | M1527094C | 样品裂解液 | 液体 | 50 mL | 2-8℃. |
| M1527094DT | 哺乳动物基因组DNA抽提试剂盒 (蛋白酶K法) (-20℃组分) | M1527094D | 蛋白酶 K 溶液 | 液体 | 500 μL | -20℃.避光 |
危险品化学品经营许可证（带存储）