计算溶液所需的质量、体积或浓度。
无内毒素BAC/PAC大型质粒大量提取试剂盒
有货
为什么选择此级别
BioReagent,分子生物学级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
室温,-20°C储存。超低温运输 。请查阅批次 COA 获取详细规格。
质量文档
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。
文献证明
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。
概述
本试剂盒用碱裂解法从培养菌中提取质粒DNA，采用独特的溶液配方和内毒素清除试剂，只需要几次简单离心去除蛋白质、多糖、内毒素、RNA等杂质，获得高质量的质粒DNA。纯化DNA的OD260/280通常在1.8左右，得到的质粒可直接应用于细胞转染甚至动物体内实验等对DNA纯度要求很高的工作中。纯化后期过程均在 Eppendorf 管中操作，方法简单，不需特殊设备，无需过柱，不用酚氯仿抽提；基本可完全回收细菌裂解释放出的质粒，不必担心质粒DNA的丢失。本方法提取纯化质粒DNA，对质粒损伤小，即使是10kb甚至100kb以上的大型质粒或超大型BAC/PAC质粒，只要碱裂解法能够提取，就可以有效纯化。
产品特点
1. 不需要使用有毒的苯酚，氯仿等试剂。快速、方便、从100-140 mL大肠杆菌LB培养液中，可快速提取0.2-0.5 mg纯净的高拷贝质粒DNA，提取率达80-90%。
2. 获得的质粒产量高，超螺旋比例高，纯度好，可直接用于酶切、转化、PCR、体外转录、测序等各种分子生物学实验。
3. 内毒素含量极低（<0.1 EU/μg DNA），可直接应用于细胞转染。
注意事项
1. 第一次使用时，将试剂盒所带全部的RNase A加入溶液P1后（终浓度100 μg/mL） 置于4℃保存。如果溶液P1中RNase A失活，提取的质粒可能会混杂有微量RNA残留，在溶液P1中补加RNase A即可。
2. 内毒素清除剂在4℃可保存一个月，如果要长期保存，建议保存在-20℃。
3. 环境温度低时溶液P2中SDS可能会析出出现浑浊或者沉淀，可在37℃水浴加热几分钟，即可恢复澄清，不要剧烈摇晃，以免形成过量的泡沫。
4. 提取的质粒量与细菌培养浓度、质粒拷贝数等因素有关。如果所提质粒为低拷贝质粒或大于10kb的大质粒，应加大菌体使用量，同时按比例增加P1、P2、PⅢ的用量。
5. 提取大质粒时操作动作要轻柔，应该使用剪大了开口的吸头，防止机械剪切对DNA的损坏。
6. DNA 沉淀液沉淀离心后，可能看不到明显沉淀。如未见沉淀，担心DNA丢失，可保留上清液，待完成全部操作后电泳鉴定，以确定是否获得终产物（数百微克DNA离心沉淀在管的侧壁上，可能无法看到明显团块）。
7. 得到的质粒DNA可用琼脂糖凝胶电泳和紫外分光光度计检测浓度与纯度。OD260值为1相当于大约50 μg/mL DNA。电泳可能为单一条带，也可能为2条或者多条DNA 条带，这主要是不同程度的超螺旋构象质粒泳动位置不一造成，与提取物培养时间长短、提取时操作剧烈程度等有关。本公司产品正常操作情况下基本超螺旋可以超过95%。
8. 本试剂盒仅限科研使用，不可用于临床诊断及其他用途。
自备试剂：异丙醇、70%乙醇
操作步骤
注意：
- 将RNase A（干粉）倒入溶液P1中，混匀，使用后置于2-8℃保存。
1. 取过夜培养菌<140 mL菌液，装入合适的离心瓶中，4,500~6,000×g于4℃离心10 min沉淀菌体，完全弃除上清。
2.加入 5 mL溶液P1，充分混悬震荡菌体沉淀，使其完全分散开，至无絮块存在。细菌悬液移入50 mL离心管中，室温放置3~5 min。
3. 加入5 mL溶液P2，轻轻颠倒离心管6~8次，室温放置5 min，使细菌完全裂解，溶液透明。
4. 加5 mL溶液PⅢ，立即颠倒离心管6~8次，充分混匀，至白色絮状物产生。冰上放置5-10 min。上述裂解液于4℃ 12,000~16,000×g离心15 min，小心吸出上清，移入新的50 mL离心管中。
5. 加入10 mL异丙醇，颠倒离心管，充分混匀（可选：室温放置10 min）。
6. 于4℃ 12,000~16,000×g 离心10 min，小心弃去上清，倒置轻轻沥干残余液体，加入3-5 mL 70%乙醇漂洗一遍，最高速离心5 min，弃上清，晾干沉淀。
7. 加入1.4 mL溶液P1完全溶解沉淀团块（可用宽口吸管轻轻吹打辅助溶解）。移入新的1.5 mL离心管中，60℃水浴放置10~20 min。
8. 最高速离心2 min，取上清转入2个新的1.5 mL离心管中（每个700 μL）。每管加入55 μL杂质清除液A，颠倒充分混匀。
9. 加入约0.1体积（约80 μL）冰预冷的内毒素清除剂，颠倒旋转7-10次（30秒左右）充分混匀，冰浴或者冰上放置≥10 min，中间偶尔颠倒混匀几次。内毒素清除剂加入上清后，上清会变得浑浊，但是冰浴后应恢复清亮。
10. 37℃水浴，溶液又会变为浑浊，颠倒混匀后37℃温育5 min。
11. 室温 16,000×g 离心5-10 min分相（温度低时，内毒素清除剂无法分相，因此必须至少20℃以上室温离心）。
12. 溶液必须分为上下两相，否则应重复步骤10-11。上层水相含DNA，下层油状相含内毒素和其它杂质。将含DNA的上层水相转移到新管（注意不要吸到油状层），弃油状层。
13. 可选步骤：根据对内毒素指标的要求重复抽提1-2次，即重复步骤9-12一两次，该步骤可以提高纯度，但是会损失一定量的质粒DNA，请根据具体需要选择使用。
14. 将上一步所得上层水相中加入等体积杂质清除液B（约750 μL），轻柔混匀后冰上放置10~30 min，4℃ 16,000×g 离心10 min，弃上清（注意不要丢失DNA），轻轻加入1 mL 70%乙醇洗涤，离心弃上清，室温倒置晾干5~10 min使乙醇完全挥发。
15. 加适量DNA溶解液（200~500 μL）溶解沉淀（可在37℃水浴中振荡以辅助溶解）。 要注意很多质粒DNA可能附着在离心管侧壁上，即使看不见，也应该充分吹打侧壁溶解回收质粒DNA。
Product Introduction
This kit uses an alkaline lysis method to extract plasmid DNA from bacterial cultures. It employs unique solution formulations and an endotoxin removal reagent. Only a few simple centrifugation steps are required to remove impurities such as proteins, polysaccharides, endotoxins, and RNA, yielding high‑quality plasmid DNA. The OD260/280 ratio of the purified DNA is typically around 1.8. The obtained plasmid can be directly used in applications requiring high DNA purity, such as cell transfection and even in vivo animal experiments. The later purification steps are carried out in Eppendorf tubes – the procedure is simple, requires no special equipment, no column purification, and no phenol/chloroform extraction. It recovers essentially all of the plasmid DNA released from bacterial lysis, so there is no need to worry about plasmid loss. This method causes minimal damage to plasmid DNA and can effectively purify even large plasmids (>10 kb) or very large BAC/PAC plasmids (>100 kb), provided they can be extracted by alkaline lysis.
Product Features
No need for toxic reagents such as phenol or chloroform. Fast and convenient: from 100–140 mL of E. coli LB culture, 0.2–0.5 mg of pure high‑copy plasmid DNA can be rapidly extracted with an extraction efficiency of 80–90%.
High yield, high proportion of supercoiled form, and high purity. The obtained plasmid can be directly used in various molecular biology experiments, including restriction enzyme digestion, transformation, PCR, in vitro transcription, and sequencing.
Extremely low endotoxin level (<0.1 EU/μg DNA), suitable for direct use in cell transfection.
Precautions
For first‑time use, add the entire vial of RNase A (powder) to Solution P1 (final concentration 100 μg/mL) and store at 4°C. If RNase A in Solution P1 becomes inactivated, trace RNA contamination may appear in the extracted plasmid – simply add additional RNase A to Solution P1.
The Endotoxin Removal Reagent can be stored at 4°C for one month. For long‑term storage, it is recommended to store at –20°C.
When ambient temperature is low, SDS in Solution P2 may precipitate, causing cloudiness or sediment. Warm the solution in a 37°C water bath for a few minutes to restore clarity. Do not shake vigorously to avoid excessive foaming.
The amount of plasmid extracted depends on factors such as bacterial culture concentration and plasmid copy number. For low‑copy plasmids or large plasmids (>10 kb), increase the amount of bacterial culture and proportionally increase the volumes of Solutions P1, P2, and PIII.
When extracting large plasmids, handle gently and use pipette tips with widened openings to avoid mechanical shearing damage to the DNA.
After centrifugation of the DNA precipitation solution, a visible pellet may not be observed. If no pellet is seen and you are concerned about DNA loss, retain the supernatant until the entire procedure is completed, then verify by electrophoresis whether the final product has been obtained (hundreds of micrograms of DNA may pellet on the side wall of the tube and may not be visible as a distinct clump).
The concentration and purity of the extracted plasmid DNA can be assessed by agarose gel electrophoresis and UV spectrophotometry. An OD260 of 1 corresponds to approximately 50 μg/mL DNA. Electrophoresis may reveal a single band, or two or more bands, which is primarily due to different migration positions of various supercoiled plasmid conformations and depends on factors such as culture duration and the vigor of operation. Under normal operating conditions with this product, the proportion of supercoiled plasmid typically exceeds 95%.
For research use only. Not for clinical diagnosis or other uses.
Materials Required but Not Provided
Isopropanol, 70% ethanol
Procedure
Note: Pour the RNase A (powder) into Solution P1, mix well, and store at 2–8°C after use.
Transfer overnight bacterial culture (<140 mL) to a suitable centrifuge bottle. Centrifuge at 4,500–6,000×g for 10 min at 4°C to pellet the cells. Remove the supernatant completely.
Add 5 mL of Solution P1. Vigorously vortex or shake to completely resuspend the cell pellet until no clumps remain. Transfer the bacterial suspension to a 50 mL centrifuge tube. Allow to stand at room temperature for 3–5 min.
Add 5 mL of Solution P2. Gently invert the tube 6–8 times and let stand at room temperature for 5 min to completely lyse the bacteria. The solution should become clear.
Add 5 mL of Solution PIII. Immediately invert the tube gently 6–8 times to mix thoroughly until a white flocculent precipitate forms. Place on ice for 5–10 min. Centrifuge the lysate at 12,000–16,000×g for 15 min at 4°C. Carefully aspirate the supernatant and transfer it to a new 50 mL centrifuge tube.
Add 10 mL of isopropanol. Invert the tube to mix thoroughly (optional: let stand at room temperature for 10 min).
Centrifuge at 12,000–16,000×g for 10 min at 4°C. Carefully discard the supernatant, invert the tube to drain residual liquid, add 3–5 mL of 70% ethanol to wash the pellet once. Centrifuge at maximum speed for 5 min, discard the supernatant, and air‑dry the pellet.
Add 1.4 mL of Solution P1 to completely dissolve the pellet (gently pipette with a wide‑bore tip to aid dissolution). Transfer to a new 1.5 mL centrifuge tube. Place in a 60°C water bath for 10–20 min.
Centrifuge at maximum speed for 2 min. Transfer the supernatant into two new 1.5 mL centrifuge tubes (700 μL each). Add 55 μL of Impurity Removal Solution A to each tube, invert to mix thoroughly.
Add approximately 0.1 volume (about 80 μL) of ice‑cold Endotoxin Removal Reagent. Invert and rotate 7–10 times (about 30 seconds) to mix thoroughly. Place on ice or in an ice bath for ≥10 min, occasionally inverting to mix. After adding the Endotoxin Removal Reagent, the supernatant will become turbid, but should become clear again after ice‑incubation.
Place in a 37°C water bath; the solution will become turbid again. Mix by inversion and incubate at 37°C for 5 min.
Centrifuge at room temperature at 16,000×g for 5–10 min to separate phases (if the temperature is low, the Endotoxin Removal Reagent will not separate phases; therefore, centrifugation must be performed at room temperature above 20°C).
The solution should separate into two phases: the upper aqueous phase contains DNA, and the lower oily phase contains endotoxins and other impurities. Transfer the upper aqueous phase (containing DNA) to a new tube (be careful not to aspirate the oily layer). Discard the oily layer.
Optional step: repeat the extraction 1–2 times according to the desired endotoxin level (i.e., repeat steps 9–12). This will increase purity but will result in some loss of plasmid DNA; choose whether to perform this step based on your specific needs.
Add an equal volume of Impurity Removal Solution B (approximately 750 μL) to the upper aqueous phase obtained in the previous step. Mix gently and place on ice for 10–30 min. Centrifuge at 16,000×g for 10 min at 4°C. Discard the supernatant (be careful not to lose the DNA pellet). Gently add 1 mL of 70% ethanol to wash. Centrifuge, discard the supernatant, and invert the tube to air‑dry for 5–10 min until the ethanol has completely evaporated.
Add an appropriate volume of DNA Dissolution Buffer (200–500 μL) to dissolve the pellet (incubate in a 37°C water bath with shaking to aid dissolution if desired). Note that much of the plasmid DNA may adhere to the wall of the centrifuge tube; even if not visible, be sure to pipette vigorously against the wall to dissolve and recover the plasmid DNA.
规格
别名
大型质粒大提试剂盒，无内毒
英文别名
BAC/PAC Mega Plasmid Isolation Kit | BAC/PAC DNA Kit
规格或纯度
BioReagent,分子生物学级
英文名称
BAC/PAC Large Plasmid Extraction Kit
应用
DNA Extraction
样品类型
质粒
储存与运输
储存条件
室温,-20°C储存
运输条件
超低温运输
稳定性与储存
各组分在相应的储存条件下保质期18个月。
组分与储存
| B1521017 | Component | 20T | Storage |
| B1521017A | RNase A（干粉） | 1 EA | RT. |
| B1521017B | 溶液P1 | 130 mL | RT. |
| B1521017C | 溶液P2 | 100 mL | RT. |
| B1521017D | 溶液PⅢ | 100 mL | RT. |
| B1521017E | 杂质清除剂A | 3 mL | RT. |
| B1521017F | 杂质清除剂B | 30 mL | RT. |
| B1521017G | 内毒素清除剂 | 10 mL | -20℃. |
| B1521017H | DNA溶解液 | 10 mL | RT. |
各组分请按照上面指示温度存放，储存18个月不影响使用效果。
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
C of A & Other Certificates(BSE/TSE, COO):
输入批号以搜索分析图谱:
通过匹配包装上的批号来查找并下载产品的 COA，每批产品都进行了严格的验证，您可放心使用！
| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 J2615487 |
分析证书 | B1521017 |
| H2603659 |
分析证书 | B1521017 |
| ZJ26F0535135 |
分析证书 | B1521017 |
技术文档和文章
溶液计算器
常见问题
本产品应如何储存？
请于?20 °C条件下保存。需冷冻保存以维持规定的有效期。
什么是「分子生物学级」？
「分子生物学级」表示该产品经过加工和检测，适用于分子生物学和蛋白质组学。典型规格包括：低或不可检测的核酸酶/蛋白酶活性、低内毒素、受控的微生物限度，以及高批间重现性。
什么是「BioReagent」？
「BioReagent」表示该产品经过加工和检测，适用于生物和生化应用。规格可能包括内毒素限值、微生物控制、无菌性、低 DNase/RNase/蛋白酶活性，以及在相关情况下的生物活性验证。
本产品如何运输？
本产品采用保温箱配冰袋运输。收货后请立即拆包，并按上述储存条件保存。
如何获取批次专用COA？
每个订单都会随附分析证书（COA），您也可以从账户订单历史中下载。如需订购前的COA，请联系您的客户代表，或使用PDP上的"申请COA"按钮——我们将提供代表性批次的COA。
需要帮助选择级别？
我们的级别选择指南涵盖了目录中所有的变体的纯度、稳定剂状态和应用适用性。
查看 BioReagent 级别指南 → 查看 Suitable for molecular biology 级别指南 →产品仅提供给经过验证的企业、机构和合格的专业人员用于研发。不适用于人类、动物、诊断或治疗。
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