计算溶液所需的质量、体积或浓度。
无内毒素高纯度质粒小量快速提取试剂盒
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。常规运输 。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
本试剂盒采用改进SDS-碱裂解法裂解细胞，粗提物通过过滤柱除去内毒素，然后离心吸附柱内的硅基质膜在高盐、低pH值状态下选择性地结合溶液中的质粒DNA，再通过去蛋白液和漂洗液将杂质和其它细菌成分去除，最后低盐、高pH值的洗脱缓冲液将纯净质粒DNA从硅基质膜上洗脱。
产品特点
1. 离心吸附柱内硅基质膜全部采用进口特制吸附膜，柱与柱之间吸附量差异极小，可重复性好。克服了国产试剂盒膜质量不稳定的弊端。
2. 独有的去蛋白液配方，可以高效去除核酸酶。即使是核酸酶含量丰富的菌株如JM系列、HB101也可以轻松去除。有效防止了质粒被核酸酶降解。
3. 独特内毒素清除方法，清除内毒素效果一般小于<0.1 EU/μg。
本试剂盒在室温储存18个月不影响使用效果。
注意事项
1. 第一次使用时，将试剂盒所带的全部RNase A加入溶液P1后（终浓度100 μg/mL）于2-8℃保存。如果溶液P1中RNase A失活，提取的质粒可能会有微量RNA残留，在溶液P1中补加RNase A即可。
2. 环境温度低时溶液P2中SDS可能会析出浑浊或者沉淀，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。
3. 避免试剂长时间暴露于空气中产生挥发、氧化、pH值变化。
4. 溶液P3和去蛋白液PD中含有刺激性化合物，操作时要戴乳胶手套，避免沾染皮肤、眼睛和衣服。若沾染皮肤、眼睛时，要用大量清水或者生理盐水冲洗。
5. 提取质粒的量与细菌培养浓度、质粒拷贝数等因素有关。一般高拷贝质粒，建议接种单菌落于1.5-4.5 mL 加合适抗生素的LB培养基，过夜培养14-16个小时，可提取出多达20µg的纯净质粒。如果所提质粒为低拷贝质粒或大于10kb的大质粒，应适当加大菌体使用量，使用5-10 mL 过夜培养物，同时按比例增加P1、P2、P3的用量，其它步骤相同。
6. 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90%。
7. 质粒DNA确切分子大小，必须酶切线性化后，对比DNA分子量Marker才可以知道。处于环状或者超螺旋的质粒，泳动位置不确定，无法通过电泳知道其确切大小。
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：无水乙醇
操作步骤
注意：
·第一次使用前请先在漂洗液WB中加入60 mL无水乙醇，充分混匀，加入后请及时标记已加入乙醇，以免多次加入！
·将RNase A（干粉）倒入溶液P1中并使用P1冲洗离心管，混匀，每次使用后置于2-8℃保存。
1. 取1.5-4.5 mL 过夜培养的菌液12,000 rpm离心30 sec，尽可能的倒干上清，收集菌体。
2. 用250 μL溶液P1重悬菌体沉淀，涡旋振荡至彻底悬浮。
3. 加250 μL的溶液P2，温和地上下翻转4-7次（裂解时间不超过5 min）使菌体充分裂解。
4. 加350 μL的溶液P3，立即温和地上下翻转4-7次，充分混匀时会出现白色絮状沉淀，12,000 rpm 离心5 min。
5. 将上清加入到过滤柱E中（过滤柱放入1.5 mL或2 mL离心管中），12,000 rpm离心2 min，收集液体。如上清量较大，请分两次离心。
6. 将上述液体转入吸附柱AC 中（吸附柱放入收集管中），12,000 rpm 离心30-60 sec，弃废液。
7. 加入500 μL去蛋白液PD，12,000 rpm离心30-60 sec，弃废液。此步骤为了去除痕量核酸酶等杂质，如所用菌株为JM系列、HB101等endA菌株或野生型菌株，核酸酶含量丰富，应加此步骤；如所用菌株为XL-1 Blue和DH5α等缺陷型菌株，核酸酶含量低，则可略过此步骤。
8. 加入500 μL漂洗液WB（请先检查是否已加入无水乙醇），12,000 rpm 离心30-60 sec弃废液。 9. 重复步骤8。
10. 将吸附柱AC放回空收集管中，12,000 rpm离心2 min，尽量除去漂洗液， 以免漂洗液中残留乙醇抑制下游反应。
11. 取出吸附柱AC，放入一个干净的离心管中，室温放几分钟。
12. 在吸附膜的中间部位加50-100 μL洗脱缓冲液EB（洗脱缓冲液事先在65-70℃水浴中加热效果更好），室温放置2 min，12,000 rpm离心1 min。如果需要较多量质粒，可将得到的溶液重新加入离心吸附柱中，离心1 min。
若用ddH₂O做洗脱液，应保证其pH值在 7.0-8.5范围内，pH值低于7.0会降低洗脱效率。洗脱缓冲液体积不应少于30 μL，体积过小影响回收效率。且DNA产物应保存在-20℃，以防DNA降解。
This kit employs a modified SDS-alkaline lysis method to lyse cells. The crude lysate is passed through a filter column to remove endotoxins. Subsequently, the silica membrane within the centrifugal spin column selectively binds plasmid DNA in the solution under high-salt, low-pH conditions. Impurities and other bacterial components are removed using a protein removal solution and a wash buffer. Finally, the purified plasmid DNA is eluted from the silica membrane with a low-salt, high-pH elution buffer.
Product Features
1.The silica membrane in the centrifugal spin column is a specially imported adsorption membrane, ensuring minimal variation in adsorption capacity between columns and excellent reproducibility. This overcomes the instability issues associated with membrane quality in domestic kits.
The unique protein removal solution formula efficiently removes nucleases. Even nuclease-rich strains such as the JM series and HB101 are easily addressed, effectively preventing plasmid degradation by nucleases.
A unique endotoxin removal method provides effective endotoxin clearance, typically resulting in less than <0.1 EU/μg.
This kit can be stored at room temperature for 18 months without affecting performance.
Precautions
For first-time use, add the entire vial of RNase A (powder) to Solution P1 (final concentration 100 μg/mL) and store at 2–8°C. If RNase A in Solution P1 becomes inactivated, trace RNA contamination may appear in the extracted plasmid. This can be remedied by supplementing Solution P1 with additional RNase A.
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.
Avoid prolonged exposure of reagents to air to prevent volatilization, oxidation, and pH changes.
Solution P3 and Protein Removal Solution PD contain irritating compounds. Wear latex gloves during operation and avoid contact with skin, eyes, and clothing. In case of contact, rinse thoroughly with plenty of water or saline.
The amount of plasmid extracted depends on factors such as bacterial culture concentration and plasmid copy number. For high-copy plasmids, inoculate a single colony into 1.5–4.5 mL of LB medium containing the appropriate antibiotic and culture overnight for 14–16 hours. This can yield up to 20 µg of pure plasmid. For low-copy plasmids or large plasmids (>10 kb), increase the bacterial amount by using 5–10 mL of overnight culture and proportionally increase the volumes of P1, P2, and P3, while keeping other steps unchanged.
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 of 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 like culture duration and the vigor of operation. Under normal operating conditions with this product, supercoiled plasmids typically exceed 90%.
To determine the exact molecular size of the plasmid DNA, it must be linearized by restriction enzyme digestion and then compared to a DNA molecular weight marker. Circular or supercoiled plasmids have variable migration positions, making it impossible to determine their exact size by electrophoresis alone.
For research use only. Not for clinical diagnosis or other uses.
Materials Required but Not Provided
Anhydrous ethanol
Procedure
Notes:
Before first use, add 60 mL of anhydrous ethanol to Wash Buffer WB. Mix thoroughly. Mark the bottle immediately after adding ethanol to avoid double addition.
Pour the RNase A (powder) into Solution P1, using P1 to rinse the vial. Mix well. Store at 2–8°C after each use.
Centrifuge 1.5–4.5 mL of overnight bacterial culture at 12,000 rpm for 30 seconds. Remove the supernatant as completely as possible to collect the bacterial pellet.
Resuspend the bacterial pellet in 250 μL of Solution P1. Vortex until thoroughly suspended.
Add 250 μL of Solution P2. Gently invert the tube 4–7 times (lysis time should not exceed 5 minutes) to lyse the cells completely.
Add 350 μL of Solution P3, and immediately and gently invert the tube 4–7 times. A white flocculent precipitate should appear upon thorough mixing. Centrifuge at 12,000 rpm for 5 minutes.
Transfer the supernatant to a Filter Column E (placed in a 1.5 mL or 2 mL centrifuge tube). Centrifuge at 12,000 rpm for 2 minutes to collect the liquid. If the supernatant volume is large, centrifuge in two separate batches.
Transfer the collected liquid to a Spin Column AC (placed in a Collection Tube). Centrifuge at 12,000 rpm for 30–60 seconds. Discard the flow-through.
Add 500 μL of Protein Removal Solution PD. Centrifuge at 12,000 rpm for 30–60 seconds. Discard the flow-through.
*This step removes trace nucleases and other impurities. It is necessary for nuclease-rich strains such as JM series, HB101 (endA+), or wild-type strains. For nuclease-deficient strains such as XL-1Blue and DH5α, this step can be omitted.*
Add 500 μL of Wash Buffer WB (ensure that anhydrous ethanol has been added). Centrifuge at 12,000 rpm for 30–60 seconds. Discard the flow-through.
Repeat step 8.
Place the Spin Column AC back into the empty Collection Tube. Centrifuge at 12,000 rpm for 2 minutes to remove residual wash buffer, which could inhibit downstream reactions due to residual ethanol.
Transfer the Spin Column AC to a clean centrifuge tube. Allow it to stand at room temperature for a few minutes.
Add 50–100 μL of Elution Buffer EB (pre-heating the buffer in a 65–70°C water bath improves efficiency) to the center of the silica membrane. Let it stand at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 1 minute. To increase the yield, reload the eluate onto the column and centrifuge again for 1 minute. If using ddH₂O for elution, ensure its pH is within the range of 7.0–8.5; pH values below 7.0 reduce elution efficiency. The elution buffer volume should not be less than 30 μL, as a smaller volume affects recovery efficiency. The DNA product should be stored at –20°C to prevent degradation.
规格
别名
无内毒高纯度快速质粒小提试剂盒 | 无内毒高纯度质粒DNA小量抽提试剂盒（离心柱法）
英文别名
Endo-Free High Pure Plasmid Mini Rapid Extraction Kit | Endo-Free High Pure Rapid Mini Plasmid Kit
规格或纯度
BioReagent,分子生物学级
英文名称
Endo-Free High Purity Rapid Plasmid Mini Kit
应用
DNA Extraction
样品类型
质粒
储存与运输
储存条件
室温
运输条件
常规运输
稳定性与储存
Store at room temperature long term (18 months).
组分与储存
| E1521050 | Component | 50T | Storage |
| E1521050A | RNase A（干粉） | 1 EA | RT. |
| E1521050B | 溶液P1 | 15 mL | RT. |
| E1521050C | 溶液P2 | 15 mL | RT. |
| E1521050D | 溶液P3 | 20 mL | RT. |
| E1521050E | 去蛋白液PD | 15 mL | RT. |
| E1521050F | 漂洗液WB | 15 mL | RT. |
| E1521050G | 过滤柱E | 1 EA×50 | RT. |
| E1521050H | 洗脱缓冲液EB | 10 mL | RT. |
| E1521050I | 吸附柱AC | 1 EA×50 | RT. |
| E1521050J | 收集管（2 mL） | 1 EA×50 | RT. |
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 |
|---|---|---|
 ZJ26F0535919 |
分析证书 | E1521050 |
技术文档和文章
溶液计算器
常见问题
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。
本产品应如何储存？
请于室温条件下保存。
本产品如何运输？
本产品按常温标准条件运输，无需温控包装。
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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