上海金畔生物科技有限公司代理New England Biolabs(NEB)酶试剂全线产品,欢迎访问官网了解更多产品信息和订购。
产品信息
(美国客户请注意:订购此产品时,您将收到两个独立包装的盒子,个盒子包含配有干冰的感受态细胞;另一个盒子包含配有蓝冰的 NEBuilder 高保真 DNA 组装预混液。)
对于大于 15 kb 组装片段的转化,NEB 推荐使用 NEB 10-beta E. coli 感受态细胞(高 效级,NEB #C3019)。使用 NEBuilder 高保真 DNA 组装预混液的用户可在购买 NEBuilder 高保真 DNA 大片段组装试剂盒时享受购买感受态细胞的折扣。
NEBuilder 高保真 DNA 组装预混液可以高效、准确地组装 DNA 片段。不管片段的长短和末端匹配性如何,该方法都可以无缝组装多个 DNA 片段。还可以组装单链寡核苷酸或含 15-30 bp 重叠区的不同片段长度的 DNA 片段。此方法操作简单、灵活,以预混液形式提供,主要用于合成生物学和多片段的一步克隆。在同一反应缓冲液中有几种不同的酶活性(见图 1):
- 核酸外切酶活性会产生单链 3´ 突出末端,促使互补的末端重复序列(重叠区)退火
- 聚合酶活性会填补退火片段的缺口
- DNA 连接酶活性能在组装后的 DNA 切刻处进行连接
最终形成双链闭合的 DNA 分子,可作为 PCR、RCA 的模板或其它分子生物学应用,包括直接转化大肠杆菌。NEBuilder 高保真 DNA 组装克隆试剂盒随附 NEB 5-alpha E.coli 感受态细胞(高效级)。
NEBuilder 高保真 DNA 组装试剂盒提供多种剂型:含 NEB 5-alpha 化学感受态细胞(克隆试剂盒,NEB #E5520),含 NEB 10-beta 化学感受态细胞(大片段组装试剂盒,NEB #E2623);以及不含感受态细胞(预混液,NEB #E2621)。NEB 5-alpha 化学感受态细胞适用于小于等于 15 kb 片段的常规组装。对于大于 15 kb 的组装,NEB 建议使用 NEB 10-beta E. coli 感受态细胞(高效级,NEB #C3019)或 NEB 10-beta E. coli 电转感受态细胞(NEB #C3020)。如果组装基因包含重复序列,应使用 NEB Stable E. coli 感受态细胞(NEB #C3040)。
NEBuilder 的应用:
- 定点突变
- sgRNA-Cas9 表达载体构建 | 视频
- 线性酵母表达框组装
为帮助您选择最佳 DNA 组装方法,请参考合成生物学/DNA 组装选择指南。
如需帮助设计用于 NEBuilder 的引物,请观看引物设计视频。
- 产品类别:
- DNA Assembly, Cloning and Mutagenesis Kits Products
- 应用:
- NEBuilder® HiFi DNA Assembly,
- Genome Editing Applications
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试剂盒组成
本产品提供以下试剂或组分:
NEB # 名称 组分货号 储存温度 数量 浓度 -
E2623S Multi-temperature NEB® 10-beta Competent E. coli (High Efficiency) C3019H -80 NEB® 10-beta/Stable Outgrowth Medium B9035SVIAL 4 1 x 25 ml Not Applicable pUC19 Vector N3041AVIAL -20 1 x 0.025 ml 50 pg/µl NEB® 10-beta Competent E. coli (High Efficiency) C3019HVIAL -80 20 x 0.05 ml Not Applicable NEBuilder® HiFi DNA Assembly Master Mix E2621S -20 NEBuilder® Positive Control N2611AVIAL -20 2 x 0.05 ml Not Applicable NEBuilder® HiFi DNA Assembly Master Mix M5520AVIAL -20 2 x 0.1 ml 2 X
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特性和用法
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优势和特性
Features
与 GeneArt Gibson 组装预混液和 In-Fusion Snap 组装预混液相比,具有更多的优势:
- 组装后保真度高、正确率高,无需反复筛选、测序。
- 更高效地连接 DNA 片段,轻松应对更大片段或较低 DNA 起始量的组装
- 由于可去除 5´ 和 3´ 末端的不匹配序列,NEBuilder 高保真产品可进行连续组装。(省去耗时的 PCR 扩增步骤,节省时间)
- 使用合成的单链 DNA 寡核苷酸桥式连接两个双链 DNA 片段(如接头插入或 gRNA 建库),构建简单又快速
- 适用于较低的 DNA 起始量要求
- NEBuilder 高保真产品可兼容使用 Gibson 组装® 预混液和 In-Fusion 组装预混液设计的片段,因此可轻松地从其它体系转用 NEBuilder 高保真产品
- 使用 NEBuilder 产品,无需缴纳授权费
超越传统克隆的优势:
- 便捷快速的无缝克隆有助于节省时间
- 一个系统既可用于“标准长度”片段的组装,也可用于大片段的组装,片段最多可达 11 个。
- 无需改动现有实验流程,DNA 可立即进行转化,或作为 PCR 或 RCA 的模板。
- 适用性强,适用于各种 DNA 改造,包括定点突变。
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相关产品
相关产品
- t1010-monarch-plasmid-miniprep-kit
- Monarch® DNA 胶回收试剂盒
- Monarch® PCR & DNA 纯化试剂盒(5 μg)
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注意事项
- 订购此试剂盒后,您将收到 3 个单独包装的产品:2X(NEB #E2621S)和 1X(NEB #C3019H)(感受态细胞单独装在一个配有干冰的盒子中)。
- NEBuilder 高保真 DNA 组装预混液和阳性对照贮存于 -20℃。NEB 10-beta/Stable Outgrowth 培养基贮存于 4℃。感受态细胞贮存于 -80℃。
- 为确保成功组装以及后续成功转化组装的 DNA,NEB 建议遵循以下做法:
DNA:如果所有 PCR 产物的总体积不超过组装反应体积的 20%,无需进行 PCR 产物纯化。PCR 产物体积过多会引入更多 PCR 产物中存在的 PCR 反应缓冲液和未使用的引物,从而降低高保真 DNA 组装和转化的效率。PCR 产物柱纯化可能会使高保真 DNA 组装和转化的效率增加 2-10 倍,在组装三个或更多 PCR 片段时,或组装长度超过 5 kb 的片段时,强烈建议进行 PCR 产物柱纯化。用于组装的纯化 DNA 可溶于 ddH2O(推荐使用 Milli-Q® 水或同等纯度的水)、TE 或其它稀释缓冲液。
- 插入片段:直接将片段组装到克隆载体中时,组装片段的浓度应比载体浓度高至少 2 倍。要将 4 个或更多片段组装到载体中,建议使用等摩尔比例的片段。
- 生物学特性:一些 DNA 结构,包括反向重复序列和串联重复序列,会被大肠杆菌选择性丢失。大肠杆菌无法耐受某些重组蛋白,因此可能会导致转化效率较低或菌落较小。
操作说明、说明书 & 用法
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操作说明
- NEBuilder HiFi DNA Assembly Reaction Protocol
- NEBuilder® HiFi DNA Assembly Chemical Transformation Protocol (E2621, E5520, E2623)
- NEBuilder Assembly of a PCR Fragment
- Protocol for cloning DNA containing repeat elements (C3040)
- Protocol for Bridging double-stranded DNA with a single-stranded DNA oligo using NEBuilder HiFi DNA Assembly (NEB #E2621)
- Protocol for assembling annealed DNA oligonucleotides and a double-stranded DNA vector using NEBuilder HiFi DNA Assembly (NEB #E2621)
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说明书
产品说明书包含产品使用的详细信息、产品配方和质控分析。- manualE2621_E5520
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使用指南
- Guidelines for using NEBuilder® HiFi DNA Assembly
- Optimization Tips for NEBuilder® HiFi DNA Assembly and NEB® Gibson Assembly
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应用实例
- Construction of an sgRNA-Cas9 expression vector via single-stranded DNA oligo bridging of double-stranded DNA fragments
- Improved method for assembly of linear yeast expression cassettes using NEBuilder® HiFi DNA Assembly Master Mix
- Bridging dsDNA with a ssDNA Oligo and NEBuilder® HiFi DNA Assembly to create an sgRNA-Cas9 Expression Vector
- Nanoliter Scale DNA Assembly Utilizing the NEBuilder® HiFi Cloning Kit with the Labcyte® Echo® 525 Liquid Handler
工具 & 资源
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选择指南
- Synthetic Biology/DNA Assembly Selection Chart
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Web 工具
- NEBioCalculator®
- NEBuilder® Assembly Tool
- NEBuilder® Protocol Calculator
FAQs & 问题解决指南
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FAQs
- I am not sure whether to choose NEBuilder HiFi DNA Assembly or NEB Gibson Assembly? How are the products different?
- What are the advantages of this method compared to traditional cloning methods?
- What is the largest single fragment that has been assembled with NEBuilder HiFi DNA Assembly Master Mix?
- How many fragments of DNA can be assembled in one reaction?
- What are the shortest overlaps that can be used with this assembly method?
- Can ≤ 200 bp dsDNA fragments be assembled by this method?
- Can multiple ssDNA oligonucleotides be assembled with dsDNA fragments?
- Can longer or shorter incubation times be used?
- Will the reaction work at other temperatures?
- Is it necessary to purify PCR products when doing DNA assemblies using either NEBuilder HiFi DNA Assembly Master Mix or Gibson Assembly Master Mix?
- Is it necessary to inactivate restriction enzymes after vector digestion?
- I would like to produce overlapping dsDNA fragments by PCR. Do I need to use PCR primers that have been purified by PAGE or HPLC?
- I would like to assemble ssDNA oligonucleotides into dsDNA fragments. Do I need to use oligonucleotides that have been purified by PAGE or HPLC?
- Can I PCR-amplify the assembled product?
- What should I do if my assembly reaction yields no colonies, a small number of colonies, or clones with the incorrect insert size following transformation into E. coli?
- How can I reduce the number of vector-only background colonies?
- Can I use electroporation instead of chemical transformation?
- Are there any differences between the requirements for 2–3 fragment assemblies versus 4+?
- Can I use PCR product amplified from Taq DNA polymerase?
- Can I Use other primer design tools such as SnapGene for Gibson Assembly, to design primers for NEBuilder HiFi DNA Assembly?
- Are there any differences between NEBuilder HIFi DNA Assembly Master Mix, the NEBuilder HiFi DNA Assembly Cloning Kit and NEBuilder HiFi DNA Assembly Bundle for Large Fragments?
- What type of competent cells are suitable for transformation of DNA constructs created using NEBuilder HiFi DNA Assembly Master Mix?
- Can I use a 15-nt overlap that is entirely composed of His-tag repeats (i.e., CACCACCACCACCAC)?
- Is this method applicable to the assembly of repetitive sequences?
- What is the difference between NEBuilder HiFi DNA Assembly Master Mix/DNA Assembly Cloning kit/Bundle for Large Fragments kit and the current NEB Gibson Assembly Master Mix/Cloning Kit?
- Can NEBuilder® HiFi DNA Assembly master mix remove both 3′ and 5′ end mismatches?
- How should fragments be prepared for assembly using NEBuilder HiFi?
- I would like to miniaturize DNA assembly with NEBuilder HiFi DNA Assembly Master Mix using the Labcyte Echo Liquid Handler. Do you have any information that I can refer to help me automate DNA assembly?
- How does overlap length affect NEBuilder HiFi DNA Assembly efficiency?
- What antibiotic resistance is encoded in the NEBuilder Positive Control (assembly control)?