Thermo Scientific CloneJET PCR 克隆试剂盒
灵活。创新。快速。
Thermo Scientific CloneJET PCR 克隆试剂盒是一款多功能的阳性克隆筛选系统,可高效克隆任何 PCR 产物。只需五分钟即可连接至阳性筛选载体,并获得超过99%的重组克隆。实验室常用的 E.coli菌株都可用该连接产物直接进行转化。
为什么使用 CloneJET PCR 克隆试剂盒?
- 快捷——可在短短5分钟内克隆 PCR 产物
- 多功能性——适用于磷酸化或非磷酸化 的DNA 片段,以及平末端和粘性末端 PCR 产物
- 高效——可获得超过99%的阳性重组克隆,且无克隆背景
- 经济——无需昂贵的蓝白斑筛选
- 兼容性强——可直接转化进常用 E. coli 菌株,包括 TOP10 和 XL1-Blue
-
阳性克隆筛选原理
Click to enlarge
pJET1.2/blunt 载体携带一个致死性限制性酶基因,此基因在 DNA 片段插入克隆位点后即被破坏。因此,只有含重组质粒的细菌细胞才能生长形成菌落。如果 pJET1.2/blunt 载体在没有片段插入的情况下重新环化,会表达致死性限制性酶,其在转化后杀死宿主 E. coli 细胞。这种阳性筛选大大加快了菌落筛选的进程,节省了蓝白斑筛选所需的额外成本。
为了便于对插入片段进行鉴定和后续处理,pJET 1.2/blunt 载体的多克隆位点包含两个 BgIII 识别序列,横跨插入位点两端。此外,载体含有用于 in vitro 和 in vivo转录以及插入片段测序的 T7 启动子。
高效
通过校正 DNA 聚合酶(例如 Phusion DNA 聚合酶)获得的平末端 PCR 产物可在5分钟内直接连接到载体中。使用 CloneJet PCR 克隆试剂盒包含的专有热稳定 DNA 平端化酶,可在连接前5分钟内将具有 3'-A 突出端的 PCR 产物进行平端化处理。CloneJET 克隆试剂盒的平末端和粘性末端片段的克隆效率,和基于连接酶的克隆试剂盒(供应商 A)相当,如图1所示。但在使用基于拓扑异构酶的克隆试剂盒(供应商 B)克隆平末端 DNA 片段时,克隆效率大幅下降。
Click to enlarge
图1.粘性末端和平末端 PCR 产物克隆效率。根据供货商推荐的方案,将由 Taq DNA 聚合酶或 Phusion DNA 聚合酶生成的976 bp PCR 产物连接到不同的克隆载体中。使用供货商指定片段作为阳性对照。各取2 μl 连接混合物转化 E. coli DH10B 细胞。供货商提供的感受态细胞用于相应基于拓扑异构酶的反应。根据对照物的阳性重组百分比,计算克隆效率。
尽管 CloneJET PCR 克隆系统的克隆效率与基于连接酶的 PCR 克隆试剂盒相当,但使用 CloneJET 试剂盒产生的重组克隆的转化效率要比后者高2到10倍(图2)。
应用
- 克隆长达10 kb 的平末端或3'-dA 加尾 PCR 产物
- 克隆由限制性内切酶酶切后的 DNA 片段
- 克隆DNA的测序
- 由T7启动子引导的克隆插入片段 in vitro 和 in vivo 转录
试剂盒组分
试剂盒中包括哪些组分?
订购信息:
Thermo Scientific™
CloneJET PCR 克隆试剂盒
货号: K1231
相关应用: PCR Cloning
技术支持客户服务
Thermo Scientific CloneJET PCR 克隆试剂盒也可用于 DH10B 细胞 (K123120),是一款先进的阳性选择系统,可高效克隆由任何热稳定 DNA 聚合酶生成的 PCR 产物。也可以克隆任何其他的钝性或黏性末端DNA片段。其非常适用于磷酸化或非磷酸化DNA片段。只需 5 分钟即可连接至所包含的阳性分选载体,并产生超过 99% 的重组克隆。将由校正酶生成的平末端 PCR 产物直接接入克隆载体。
在连接前 5 分钟内使用试剂盒提供的热稳定 DNA 平末端化酶将由非校正 DNA 聚合酶或 DNA 聚合酶混合物生成的 PCR 产物处理成平末端。所有常见的实验室大肠杆菌菌株都可以使用连接产物直接进行转化。
特点
CloneJET PCR 克隆试剂盒中包含一种新的即用型阳性选择克隆载体 pJET1.2/blunt。该载体中有一个致命型限制性酶基因,此基因在 DNA 插入片段接入克隆位点后即被破坏。因此,只有含重组质粒的细菌细胞才能形成菌落。不含插入片段的再循环 pJET1.2/blunt 载体分子会表达致死性限制性内切酶,继而在转化后杀死宿主大肠杆菌细胞。此阳性选择大大加快了菌落筛选的进程,节省了蓝白斑筛选所需的额外成本。
为了便于对插入片段进行映射和处理,pJET1.2/blunt 克隆载体的多克隆位点包含两个 BglII 识别序列,位于插入位点两侧。此外,载体含有用于体外和体内转录以及插入片段测序的 T7 启动子。
产品优势
•快速—在仅 5 分钟内完成 PCR 克隆
•较高效率–>99% 阳性克隆
•无克隆背景—阳性选择载体
•多用途—是平末端或黏性末端克隆的理想选择
•经济—无需进行昂贵的蓝白斑筛选
应用
•克隆高达 10 kb 的平末端或 '3-dA 尾 PCR 产物
•克隆限制性内切酶生成的 DNA 片段
•克隆 DNA 测序
•从 T7 启动子体外和体内转录克隆插入片段
包括
•pJET1.2/blunt 克隆载体
• T4 DNA 连接酶
• 2X 反应缓冲液
• DNA 平末端化酶
• pJET1.2 正向测序引物 (5'-CGACTCACTATAGGGAGAGCGGC-3'
)
• pJET1.2 反向测序引物 (5'-AAGAACATCGATTTTCCATGGCAG-3'
)
• 对照 PCR 产物
• 无核酸酶水
•详细方案
电穿孔前,始终使用 GeneJET PCR 纯化试剂盒 #K0701 对连接混合物进行柱纯化或使用氯仿进行提取。混合物中存在蛋白质和盐分会抑制电穿孔。
相关产品
CloneJET PCR 克隆试剂盒
快速 DNA 末端修复试剂盒
规格
耐抗生素细菌
氨苄青霉素 (AmpR)
细菌或酵母菌株
不包括在内
克隆方法
限制性内切酶 ⁄ MCS,平末端 PCR
适用于(应用)
PCR 克隆
反应次数
20次反应
产品线
CloneJET
产品类型
PCR 克隆试剂盒
载体
pJET1.2/blunt
产品规格
试剂盒
Thermo Scientific Competent Cells
Confidently competent
Thermo Scientific DH10B, DH5α, and BL21(DE3) competent cells are produced with stringent quality control to enable reliable, consistent performance in cloning workflows. Thermo Scientific DH10B competent cells are also included in the Thermo Scientific CloneJET PCR Cloning Kit and in the Thermo Scientific Phusion Site-Directed Mutagenesis Kit.
Which competent cells are best for my application?
|
Thermo Scientific DH10B Competent Cells Order |
Thermo Scientific DH5α Competent Cells Order |
Thermo Scientific DH5α Competent Cells for Subcloning Order |
Thermo Scientific BL21(DE3) Competent Cells Order |
Quantity |
20 reactions 10 x 100 µL |
20 reactions 10 x 100 µL |
40 reactions 4 x 500 µL |
20 reactions 10 x 100 µL |
Primary applications |
- cDNA library
- Gene bank construction
- Cloning large plasmids and BACs
- Site-directed mutagenesis
|
- Subcloning DNA and cDNA
- Plasmid isolation
|
|
|
Transformation efficiency (cfu/µg pUC19 DNA) |
> 1x109 |
> 1x109 |
> 1x106 |
> 1x107 |
Notes |
Not suitable for preparing unmethylated DNA |
Not suitable for preparing unmethylated DNA |
Not suitable for preparing unmethylated DNA |
Not suitable for maintenance of expression plasmids |
Genotype |
F– mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ (ara-leu)7697 galU galK λ– rpsL(StrR) nupG |
F– φ80lacZDM15 D(lacZYA-argF) U169 recA1 endA1 hsdR17 (rk–, mk+) phoA supE44 l– thi–1 gyrA96 relA1 |
F– φ80lacZDM15 D(lacZYA-argF) U169 recA1 endA1 hsdR17 (rk–, mk+) phoA supE44 l– thi–1 gyrA96 relA1 |
F– ompT hsdSB (rB-mB-) gal dcm (DE3) |
If you are looking for competent cells for specialized applications, view our Invitrogen competent cells here.
Thermo Scientific competent cell combination kits
Thermo Scientific CloneJET PCR Cloning Kit with DH10B
20 or 40 reactions
Order
Phusion Site-Directed Mutagenesis Kit with DH10B
20 reactions
Order
Achieve high transformation efficiencies with Thermo Scientific competent cells
Thermo Scientific DH10B competent cells
The transformation efficiencies of DH10B competent cells (Thermo Fisher Scientific), 10-beta competent E. coli I (NEB), and E. cloni 10G cells (Lucigen Corp) were compared using pUC19 DNA and following the manufacturers’ recommended protocols. The transformation efficiency specification for DH10B competent cells is 1.0 x 109 CFU/µg DNA (represented by the red dashed line). The values shown are the averages of five transformations. Error bars represent standard deviations.
Thermo Scientific DH5α competent cells
The transformation efficiencies of DH5α competent cells and 5-alpha competent E. coli (NEB) were compared using pUC19 DNA and following the manufacturers’ recommended protocols. The transformation efficiency specification for DH5α competent cells is 1.0 x 109 CFU/µg DNA (represented by the red dashed line). The values shown are the averages of five transformations.
Thermo Scientific DH10B competent cell transformation efficiency in the CloneJET PCR cloning workflow
A 976 bp PCR product was generated using either Thermo Scientific DreamTaq Green PCR Master Mix (2X) (represented by grey bar) or Thermo Scientific Phusion Hot Start II DNA Polymerase (represented by blue bar) and ligated into a cloning vector according to the manufacturers’ protocols. The transformation efficiency obtained with the control fragment provided with the CloneJET PCR Cloning Kit is represented by the yellow bar. Ligation mixtures were used to transform Thermo Scientific DH10B cells. The expected transformation efficiency of competent cells is >1 x 107 CFU/µg DNA. The transformation efficiency specification of DH10B competent cells with pUC19 DNA is 1 x 109 CFU/µg DNA. The values shown are the average of five transformations. Error bars represent standard deviations.
Efficient recovery of mutants using Thermo Scientific DH10B competent cells
The Thermo Scientific Phusion Site-Directed Mutagenesis Kit has been tested using the control plasmid and control primer mix provided in the kit, in conjunction with Thermo Scientific DH10B competent cells. The primer mix reverts the mutated internal stop codon to a functional codon; a successful mutagenesis reaction results in blue colonies on ampicillin agar plates containing X-Gal and IPTG. A high rate of mutagenesis and high transformation efficiency means that numerous mutants can be directly subjected to sequencing, eliminating additional analysis steps.
Thermo Scientific™
DH10B Competent Cells
货号: EC0113
技术支持客户服务
|
货号 |
单位规格 |
价格(CNY) |
库存状况 |
数量 |
|
EC0113 |
10 x 100 µL |
1,355.00 您的价格 登录 |
*** |
Thermo Scientific DH10B Competent Cells are high efficiency, chemically competent E. coli cells. The DH10B strain is suitable for cloning DNA containing methylcytosine, 5-hydroxymethylcytosine, and methyladenine, allowing both prokaryotic and eukaryotic genomic DNA to be cloned efficiently. These cells are ideal for the construction of gene banks or for the generation of cDNA libraries using plasmid-derived vectors. The φ80dlacZΔM15 marker provides α-complementation of the β-galactosidase gene from pUC or similar vectors to allow blue/white colony screening on bacterial agar plates containing Bluo-Gal or X-Gal.
• High transformation efficiency: >1x109 cfu/µg pUC19 DNA
• Suitable for routine and high-throughput cloning applications
• Allows for blue/white colony screening
• Convenient two reactions per vial packaging
• S.O.C. Outgrowth medium included
Applications
DH10B Competent Cells are suitable for variety of applications requiring high transformation efficiency:
• Efficient DNA cloning both of prokaryotic and eukaryotic origin
• Cloning of large plasmids and BACs
• Ideal for generation of cDNA libraries using plasmid-derived vectors
• Site directed mutagenesis
Genotype
F– mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ (ara-leu)7697 galU galK λ– rpsL(StrR) nupG
Note: Optimized for use with the CloneJET PCR Cloning Kit (Cat. Nos. K1231, K1232) and Phusion Site-Directed Mutagenesis Kit (Cat. No. F541), and available as combo versions (Cat. Nos. K123120, K123240, F542).
规格
细菌或酵母菌株
DH10B™
蓝色/白色筛查
Yes
适用于(应用)
Cloning
高通量能力
Not High-throughput Compatible
产品线
Thermo Scientific™
扩增 ccdB 载体
Not for ccdB vector propagation
数量
10 x 100 μL
运输条件
Dry Ice
产品规格
Tube
产品类型
Competent Cells
种属
E. coli
是否含 F' 附加体
Lacks F' Episome
细胞类型
Chemically Competent
是否可克隆甲基化 DNA
Yes
内容与储存
• 10 x DH10B Competent Cells (100 µL)
• pUC19 DNA (50 µL) (10 pg/µL)
• S.O.C. Medium (5 mL)
Store at -80°C.
Thermo Scientific™
DH5α 感受态细胞
货号: EC0112
技术支持客户服务
|
货号 |
单位规格 |
价格(CNY) |
库存状况 |
数量 |
|
EC0112 |
10 x 100 µL |
1,164.00 您的价格 登录 |
*** |
Thermo Scientific DH5α 感受态细胞是高效、化学感受态大肠杆菌细胞,适用于使用质粒衍生载体构建基因库或生成 cDNA 文库。φ80dlacZΔM15 标志物提供了来自 pUC 或类似载体的 β-半乳糖苷酶基因的α-互补 ,可用于在含有 Bluo-Gal 或 X-Gal 的细菌琼脂平板上进行蓝白斑菌落筛选。DH5α 细胞中的 RecA1 和 endA1 突变可提高提取质粒 DNA 的插入片段稳定性和质量。
•高转化效率:>1x109 cfu/µg pUC19 DNA
• 适用于常规克隆和高通量克隆应用
• 可进行蓝白斑菌落筛选的基因标志物
• 便利 每瓶包装两次反应
• S.O.C.包含外生长培养基
应用
DH5α 感受态细胞,适用于需要高转化效率的多种应用:
•来自 PCR 的高效 DNA 克隆,cDNA 生成反应
• recA1 标志物有助于轻松转化 DNA
• 适用于使用质粒衍生载体生成 cDNA 文库
• 定点诱变
基因型
F– φ80lacZΔ M15 Δ (lacZYA-argF) U169 recA1 endA1 hsdR17 (rK– mK+) phoA supE44 λ- thi–1 gyrA96 relA1
规格
细菌或酵母菌株
DH5α™
蓝色/白色筛查
是
适用于(应用)
Cloning
高通量能力
不兼容高通量应用
产品线
Thermo Scientific™
扩增 ccdB 载体
不适合扩增ccdB载体
数量
10 x 100 μL
运输条件
干冰
产品规格
管
产品类型
Competent Cells
种属
大肠杆菌
是否含 F' 附加体
缺少 Fin 附加体
细胞类型
化学感受态
是否可克隆甲基化 DNA
否
内容与储存
• 10 x DH5α 感受态细胞 (100 µL)
• pUC19 DNA (50 µL) (10 pg/µL)
• S.O.C.培养基 (5 mL)
在 -80°C 下储存。
Thermo Scientific™
亚克隆用 DH5α 感受态细胞
货号: EC0111
技术支持客户服务
|
货号 |
单位规格 |
价格(CNY) |
库存状况 |
数量 |
|
EC0111 |
4 x 500 µL |
544.00 您的价格 登录 |
*** |
亚克隆用 Thermo Scientific DH5α 感受态细胞是化学感受态大肠杆菌细胞,推荐用于常规亚克隆至质粒载体中。Subcloning Efficiency 细胞不适用于生成 cDNA 文库。φ80dlacZΔM15 标志物提供了来自 pUC 或类似载体的 β-半乳糖苷酶基因的 α-互补,可用于在含有 Bluo-Gal 或 X-Gal 的细菌琼脂平板上进行蓝白斑菌落筛选。
•转化效率:>1x106 cfu/µg pUC19 DNA
• 适用于常规克隆应用
•便利的经济包装
• 包含可进行蓝白斑菌落筛选的基因标志物
应用
亚克隆用 DH5α 感受态细胞适用于标准 DNA 克隆应用:
•PCR 或限制性酶切反应后的 DNA 克隆
• 亚克隆(将 DNA 从亲本载体转移至目的载体)
• 质粒分离
基因型
F– φ80lacZΔ M15 Δ (lacZYA-argF) U169 recA1 endA1 hsdR17 (rK– mK+) phoA supE44 λ- thi–1 gyrA96 relA1
规格
细菌或酵母菌株
DH5α™
蓝色/白色筛查
是
适用于(应用)
Cloning
高通量能力
不兼容高通量应用
产品线
Thermo Scientific™
扩增 ccdB 载体
不适合扩增ccdB载体
数量
4 x 500 μL
运输条件
干冰
产品规格
管
产品类型
Competent Cells
种属
大肠杆菌
是否含 F' 附加体
缺少 Fin 附加体
细胞类型
化学感受态
是否可克隆甲基化 DNA
否
内容与储存
亚克隆用 • 4 x DH5α 感受态细胞 (500 µL)
• pUC19 DNA (50 µL) (100 pg/µL)
在 -80°C 下储存。
Thermo Scientific™
BL21(DE3) 感受态细胞
货号: EC0114
技术支持客户服务
Thermo Scientific BL21(DE3) 感受态细胞适用于无毒异源基因的表达。该菌株含有携带受 lacUV5 启动子控制的 T7 RNA 聚合酶基因的 λ DE3 前噬菌体,实现 IPTG 诱导的 T7 RNA 聚合酶表达。BL21(DE3) 是一种大肠杆菌 B 菌株,不含 lon 蛋白酶。其也缺乏外膜蛋白酶 OmpT。不存在这两种关键蛋白酶可降低细胞中表达的异源蛋白的降解。
•高转化效率:>1x107 cfu/µg pUC19 DNA
• 专门构建用于高水平重组蛋白表达
•尽可能减少 RNA 和蛋白降解的基因标志物
•每瓶包装可方便地进行两次反应
• S.O.C.外生长培养基包括
应用
BL21(DE3) 感受态细胞适用于高水平重组蛋白的 T7 表达。
基因型
F– ompT hsdSB (rB–, mB–) gal dcm (DE3)
注:不适用于维持表达质粒。使用不携带 T7 RNA 聚合酶基因的 DH10B 或 DH5α 感受态细胞进行质粒 DNA 增殖(货号EC0112、EC0113)。
规格
细菌或酵母菌株
BL21(DE3)
蓝色/白色筛查
否
产品线
Thermo Scientific™
数量
10 x 100 μL
运输条件
干冰
产品规格
管
产品类型
Competent Cells
种属
大肠杆菌
细胞类型
化学感受态
内容与储存
• 10 x BL21(DE3) 感受态细胞 (100 µL)
• pUC19 DNA (50 µL) (10 pg/µL)
• S.O.C.培养基 (5 mL)
在 -80°C 下储存。
Thermo Scientific™
CloneJET™ PCR Cloning Kit with DH10B Competent Cells
货号: K123120
技术支持客户服务
This package includes the CloneJET PCR Cloning Kit (20 or 40 reactions) and DH10B Competent Cells that are optimized for use with the kit and can be directly transformed with the ligation product.
CloneJET PCR Cloning Kit
The Thermo Scientific CloneJET PCR Cloning Kit is an advanced positive-selection system for high-efficiency cloning of PCR products generated with any thermostable DNA polymerase. Any blunt or sticky-end DNA fragment can be cloned. The kit works well with both phosphorylated and non-phosphorylated DNA fragments. Ligation into the included positive-selection vector takes only five minutes, yielding more than 99% recombinant clones. Blunt-ended PCR products generated with a proofreading enzyme are ligated directly into the cloning vector.
PCR products generated either with non-proofreading DNA polymerases or mixtures of DNA polymerases need to be blunted prior to ligation in a five minute reaction with the thermostable DNA Blunting Enzyme provided with the kit. DH10B Competent Cells are optimized for use with the CloneJET PCR Cloning Kit and can be directly transformed with the ligation product.
The CloneJET PCR Cloning Kit contains the novel, ready-to-use positive selection cloning vector pJET1.2/blunt. The vector contains a lethal restriction enzyme gene that is disrupted by ligation of a DNA insert into the cloning site. As a result, only bacterial cells with recombinant plasmids are able to form colonies. Re-circularized pJET1.2/blunt vector molecules lacking an insert express a lethal restriction enzyme, which kills the host E. coli cell after transformation. This positive selection drastically accelerates the process of colony screening and eliminates additional costs required for blue/white selection.
• Fast—PCR cloning in only five minutes
• Highest efficiency—>99% of positive clones
• No cloning background—positive selection vector
• Versatile—ideal for blunt-end or sticky-end cloning
• Economical—no expensive blue/white screening
Applications include:
• Cloning of blunt-end or 3' dA-tailed PCR products up to 10 kb
• Cloning of DNA fragments generated by restriction enzymes
• Sequencing of cloned DNA
• in vitro and in vivo transcription of cloned inserts from the T7 promoter
Learn more about the CloneJET PCR Cloning Kit ›
DH10B Competent Cells
Thermo Scientific DH10B Competent Cells are high efficiency, chemically competent E.coli cells. The DH10B strain is suitable for cloning DNA containing methylcytosine and methyladenine, allowing both prokaryotic and eukaryotic genomic DNA to be cloned efficiently. These cells are ideal for the construction of cDNA libraries using plasmid-derived vectors. Genotype: F– mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ (ara-leu)7697 galU galK λ– rpsL(StrR) nupG.
• High transformation efficiency: >1x109 cfu/µg pUC19 DNA
• Suitable for routine and high-throughput cloning applications
• Convenient two reactions per vial packaging
• S.O.C. Outgrowth medium included
Related products
CloneJET PCR Cloning Kit without competent cells
aLICator LIC Cloning and Expression System
规格
耐抗生素细菌
Ampicillin (AmpR)
细菌或酵母菌株
DH10B
细胞类型
DH10B
克隆方法
Blunt PCR, Restriction Enzyme ⁄ MCS
效率
≥1 x 10^9
适用于(应用)
PCR Cloning
反应次数
20
产品线
Thermo Scientific™
产品类型
PCR Cloning Kit
运输条件
Dry Ice
载体
pJET1.2/blunt
产品规格
Kit
内容与储存
• 10 x DH10B Competent Cells for Subcloning (100 µL)
• pUC19 DNA (20 µL) (100 pg/µL)
• S.O.C. Medium (5 mL)
• pJET1.2/blunt Cloning Vector (24 µL) (50 ng/µL)
• 2X Reaction Buffer (300 µL)
• T4 DNA Ligase (24 µL) (5 U/µL)
• DNA Blunting Enzyme (24 µL)
• DNA Blunting Enzyme (24 µL)
• pJET1.2 Forward Sequencing Primer, 10 µM aqueous solution (50 µL)
• pJET1.2 Reverse Sequencing Primer, 10 µM aqueous solution (50 µL)
• Control PCR Product, 976 bp with 3’-dA overhangs (8 µL) (24 ng/µL)
• Water, nuclease-free, (1.25 mL)
Thermo Scientific™
Phusion™ Site-Directed Mutagenesis Kit with DH10B Competent Cells
货号: F542
技术支持客户服务
|
货号 |
单位规格 |
价格(CNY) |
|
F542 |
20 reactions |
联系我们 › |
This package includes a Phusion Site-Directed Mutagenesis Kit and DH10B Competent Cells that are optimized for use with the kit.
Phusion Site-Directed Mutagenesis Kit
The Thermo Scientific Phusion Site-Directed Mutagenesis Kit is a versatile and efficient tool for introducing point mutations, insertions, or deletions into any type of plasmid DNA. With this kit the entire plasmid is amplified using phosphorylated primers that introduce the desired changes. The amplified, linear PCR product, containing the desired mutation, is circularized in a 5-minute ligation reaction with T4 DNA Ligase. The resulting plasmid can be then transformed into the E.coli cells. DH10B Competent Cells are recommended for use with the kit.
The optimal annealing temperature for Phusion DNA polymerases may differ significantly from that of Taq-based polymerases. For optimal results start by accurately calculating your Tm with our Tm calculator.
• Robust and reliable exponential amplification method
• No requirements, such as special vectors, restriction sites, or methylation status for the target plasmid
• No need to destroy the starting template in a separate step
• Phusion Hot Start II High Fidelity DNA Polymerase minimizes unwanted secondary mutations
• Amplification of large plasmids up to 10 kb
• Hot start modification of the polymerase prevents amplification of non-specific products and unwanted degradation of primers prior to first cycle of PCR
• T4 DNA Ligase included in the kit; no purification steps before or after ligation
DH10B Competent Cells
Thermo Scientific DH10B Competent Cells are high efficiency, chemically competent E.coli cells. The DH10B strain is suitable for cloning DNA containing methylcytosine and methyladenine, allowing both prokaryotic and eukaryotic genomic DNA to be cloned efficiently. These cells are ideal for the construction of cDNA libraries using plasmid-derived vectors. Genotype: F– mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ (ara-leu)7697 galU galK λ– rpsL(StrR) nupG.
• High transformation efficiency: >1x109 cfu/µg pUC19 DNA
• Suitable for routine and high-throughput cloning and mutagenesis applications
• Convenient two reactions per vial packaging
• S.O.C. Outgrowth medium included
Related products
Thermo Scientific Phusion Site-Directed Mutagenesis Kit without competent cells
规格
细胞类型
Competent Cell
效率
≥1 x 10^9
适用于(应用)
Cellular Imaging
诱变能力
Single-Site Mutagenesis
反应次数
20
产品线
Phusion, Thermo Scientific™
产品类型
Site-Directed Mutagenesis Kit
运输条件
Dry Ice
产品规格
Kit
aLICator LIC Cloning & Expression System
Ligation Independent Cloning (LIC) is an alternative to restriction enzyme/ligase cloning and ensures high cloning efficiencies of more than 95%
The LIC method uses T4 DNA polymerase to create specific 14 to 21 nucleotide single-stranded overhangs on vector and DNA insert. T4 DNA polymerase has two enzymatic activities: 5'→3' polymerase activity and 3'→5' exonuclease activity. The exonuclease activity removes nucleotides from the 3' ends of the DNA while the polymerase activity restores the chain using dNTPs and the complementary DNA strand as a template. In the LIC protocol, only dGTP is included in the reaction, causing the 3'→5'-exonuclease and 5'→3'-polymerase activities to equilibrate at the first occurrence of cytosine in the complementary strand (see Figure 1). After annealing, the LIC vector and insert are transformed into competent E. coli cells. Covalent bond formation at the vector-insert junctions occurs within the cell to yield circular plasmid.
Successful cloning & robust protein expression
The Thermo Scientific aLICator LIC Cloning and Expression System is designed for fast and efficient LIC cloning and subsequent tight regulation of gene expression in E. coli.
pLATE bacterial expression vectors (see Figure 2) are designed for high levels of target protein expression in concert with minimized basal (uninduced) expression.
The aLICator system consists of 4 kits and 2 sets, based on the pLATE series of bacterial expression vectors. For proteins with a known preference for either the N- or C-terminal 6xHis-tag position, using the appropriate N- or C-terminal kit is recommended. When the protein structure and features are not well known, it is recommended to use an aLICator set. Sets allow cloning into three different vectors to determine the most compatible vector for further experiments. Following protein affinity purification, aminoterminal tags can be removed either via Enterokinase (EK), DDDDK^ or a novel protease: WELQut (WQ), WELQ^.
Figure 2: pLATE expression vectors use elements from bacteriophage T7 to control expression of heterologous genes in E. coli. The expression of the gene of interest is driven by a strong bacteriophage T7 promoter that is specifically recognized by T7 RNA polymerase. To express the gene of interest, E. coli strains such as BL21 (DE3), HMS 174 (DE3) must be used, in which expression of T7 RNA polymerase gene is under the control of an inducible promoter such as lacUV5. After IPTG induction, theT7 RNA polymerase is expressed within the cell, and begins transcription of genes under the T7 promoter.
aLICator highlights
- High efficiency LIC cloning
- Tight control of gene expression
- High yield expression
- Versatile – tagged or untaged protein expression with tag removal option
aLICator applications
- Directional PCR product cloning
- Tightly regulated protein expression
- Expression of toxic genes
aLICator highlights
- High efficiency LIC cloning
- Tight control of gene expression
- High yield expression
- Versatile – tagged or untaged protein expression with tag removal option
aLICator applications
- Directional PCR product cloning
- Tightly regulated protein expression
- Expression of toxic genes
aLICator LIC cloning products
|
Product |
Vector |
Catalog # |
Size |
aLICator LIC Cloning and Expression Kits |
aLICator LIC Cloning and Expression Kit 1 (untagged) |
pLATE 11 |
K1241 |
20 rxns |
aLICator LIC Cloning and Expression Kit 2 (N-terminal His-tag/EK) |
pLATE 51 |
K1251 |
20 rxns |
aLICator LIC Cloning and Expression Kit 3 (C-terminal His-tag) |
pLATE 31 |
K1261 |
20 rxns |
aLICator LIC Cloning and Expression Kit 4 (N-terminal His-tag/WQ) |
pLATE 52 |
K1281 |
20 rxns |
aLICator LIC Cloning and Expression Sets |
aLICator LIC Cloning and Expression Set 1 (All-in-One/EK) |
pLATE 11, 31, & 51 |
K1271 |
30 rxns |
aLICator LIC Cloning and Expression Set 2 (All-in-One/WQ) |
pLATE 11, 31, & 52 |
K1291 |
30 rxns |
|
WELQut Protease |
— |
EO0861 |
500 U |
Vector sequences
Note: right click and "Save link as" each file.
pLATE bacterial expression vector sequences
Control PCR fragments
仅供研究使用,不可应用于诊断程序。
aLICator LIC cloning products
|
Product |
Vector |
Catalog # |
Size |
aLICator LIC Cloning and Expression Kits |
aLICator LIC Cloning and Expression Kit 1 (untagged) |
pLATE 11 |
K1241 |
20 rxns |
aLICator LIC Cloning and Expression Kit 2 (N-terminal His-tag/EK) |
pLATE 51 |
K1251 |
20 rxns |
aLICator LIC Cloning and Expression Kit 3 (C-terminal His-tag) |
pLATE 31 |
K1261 |
20 rxns |
aLICator LIC Cloning and Expression Kit 4 (N-terminal His-tag/WQ) |
pLATE 52 |
K1281 |
20 rxns |
aLICator LIC Cloning and Expression Sets |
aLICator LIC Cloning and Expression Set 1 (All-in-One/EK) |
pLATE 11, 31, & 51 |
K1271 |
30 rxns |
aLICator LIC Cloning and Expression Set 2 (All-in-One/WQ) |
pLATE 11, 31, & 52 |
K1291 |
30 rxns |
|
WELQut Protease |
— |
EO0861 |
500 U |
Vector sequences
Note: right click and "Save link as" each file.
pLATE bacterial expression vector sequences
Control PCR fragments
仅供研究使用,不可应用于诊断程序。
Thermo Scientific aLICator™ LIC Cloning and Expression System is designed for fast and efficient ligation independent cloning and tight regulation of gene expression in E. coli. The pLATE bacterial expression vectors are designed for high levels of target protein expression in concert with minimal background (uninduced) expression, which permits expression of proteins that are toxic to E. coli cells. To streamline and facilitate the process of insert cloning into the expression vector, the aLICator system uses directional LIC cloning technology, a rapid procedure that provides high-cloning efficiencies.
The tightly regulated expression and fast, efficient directional cloning makes the aLICator LIC Cloning and Expression System the best choice for routine and toxic gene cloning and expression in E. coli.
Highlights
• High efficiency LIC cloning
• Tight control of gene expression
• High yield expression
• Versatile—tagged or untaged protein expression with tag removal option
Applications
• Directional PCR product cloning
• Tightly regulated protein expression
• Expression of toxic genes
Principle
The aLICator LIC cloning system uses directional LIC cloning technology to streamline and facilitate cloning into an expression vector. LIC ensures high-cloning efficiencies of more than 95% and eliminates the need for ligation and restriction enzyme digestion steps.
The LIC method uses T4 DNA polymerase to create specific 14 to 21 nucleotide single-stranded overhangs on the pLATE vectors and DNA inserts. T4 DNA polymerase has two enzymatic activities: 5'→3' polymerase activity and 3'→5' exonuclease activity. The exonuclease activity removes nucleotides from the 3' ends of the DNA while the polymerase activity restores the chain using dNTPs and the complementary DNA strand as a template. In the LIC protocol, only dGTP is included in the reaction, causing the 3'→5'-exonuclease and 5'→3'-polymerase activities to equilibrate at the first occurrence of cytosine in the complementary strand. After annealing, the LIC vector and insert are transformed into competent E. coli cells without the use of T4 DNA ligase. Covalent bond formation at the vector-insert junctions occurs within the cell to yield circular plasmid.
Features
The system consists of four kits based on the pLATE series of bacterial expression vectors:
• aLICator™ LIC Cloning and Expression Kit 1 - pLATE11 vector, untagged protein expression.
• aLICator™ LIC Cloning and Expression Kit 2 (N-terminal His-tag/EK)—pLATE51 vector, N-terminal His-tag protein expression.
• aLICator™ LIC Cloning and Expression Kit 3 (C-terminal His-tag)—pLATE31 vector, C-terminal His-tag protein expression.
• aLICator™ LIC Cloning and Expression Kit 4 (N-terminal His-tag/WQ)—pLATE 52 vector, N-terminal His-tag protein expression, WELQut cleavage.
• aLICator™ LIC Cloning and Expression Set 1 (All-in-One/EK)—pLATE11, pLATE51 and pLATE31 vectors, choice of untagged, N- or C-terminal His-tag protein expression.
• aLICator™ LIC Cloning and Expression Set 2 (All-in-One/WQ)—pLATE11, pLATE52, and pLATE31 vectors, choice of untagged, N- or C-terminal His tag protein expression, WELQut cleavage.
For proteins with a known preference for either the N- or C-terminal 6xHis-tag position, using the appropriate N- or C-terminal kit is recommended. When the protein structure and features are not well known, it is recommended to clone into all three vectors and determine the most compatible vector for further research.
Related Products
aLICator LIC Cloning and Expression Kit 2 (N-terminal His-tag/EK)
aLICator LIC Cloning and Expression Kit 3 (C-terminal His-tag)
aLICator LIC Cloning and Expression Set 1 (All-in-One/EK)
aLICator LIC Cloning and Expression Kit 4 (N-terminal His-tag/WQ)
aLICator LIC Cloning and Expression Set 2 (All-in-One/WQ)
规格
耐抗生素细菌
Ampicillin (AmpR)
克隆方法
LIC Cloning
适用于(应用)
PCR Cloning
反应次数
20
产品线
aLICator
产品类型
Cloning and Expression Kit
促进剂
T7
蛋白标记
Untagged
载体
pLATE
产品规格
Kit
Thermo Scientific™
aLICator LIC Cloning and Expression Kit 2 (N-terminal His-tag/EK)
货号: K1251
相关应用: PCR Cloning
技术支持客户服务
Thermo Scientific aLICator™ LIC Cloning and Expression System is designed for fast and efficient ligation independent cloning and tight regulation of gene expression in E. coli. The pLATE bacterial expression vectors are designed for high levels of target protein expression in concert with minimal background (uninduced) expression, which permits expression of proteins that are toxic to E. coli cells. To streamline and facilitate the process of insert cloning into the expression vector, the aLICator system uses directional LIC cloning technology, a rapid procedure that provides high-cloning efficiencies.
The tightly regulated expression and fast, efficient directional cloning makes the aLICator LIC Cloning and Expression System the best choice for routine and toxic gene cloning and expression in E. coli.
Highlights
• High efficiency LIC cloning
• Tight control of gene expression
• High yield expression
• Versatile—tagged or untaged protein expression with tag removal option
Applications
• Directional PCR product cloning
• Tightly regulated protein expression
• Expression of toxic genes
Principle
The aLICator LIC cloning system uses directional LIC cloning technology to streamline and facilitate cloning into an expression vector. LIC ensures high-cloning efficiencies of more than 95% and eliminates the need for ligation and restriction enzyme digestion steps.
The LIC method uses T4 DNA polymerase to create specific 14 to 21 nucleotide single-stranded overhangs on the pLATE vectors and DNA inserts. T4 DNA polymerase has two enzymatic activities: 5'→3' polymerase activity and 3'→5' exonuclease activity. The exonuclease activity removes nucleotides from the 3' ends of the DNA while the polymerase activity restores the chain using dNTPs and the complementary DNA strand as a template. In the LIC protocol, only dGTP is included in the reaction, causing the 3'→5'-exonuclease and 5'→3'-polymerase activities to equilibrate at the first occurrence of cytosine in the complementary strand. After annealing, the LIC vector and insert are transformed into competent E. coli cells without the use of T4 DNA ligase. Covalent bond formation at the vector-insert junctions occurs within the cell to yield circular plasmid.
Features
The system consists of four kits based on the pLATE series of bacterial expression vectors:
• aLICator™ LIC Cloning and Expression Kit 1 - pLATE11 vector, untagged protein expression.
• aLICator™ LIC Cloning and Expression Kit 2 (N-terminal His-tag/EK)—pLATE51 vector, N-terminal His-tag protein expression.
• aLICator™ LIC Cloning and Expression Kit 3 (C-terminal His-tag)—pLATE31 vector, C-terminal His-tag protein expression.
• aLICator™ LIC Cloning and Expression Kit 4 (N-terminal His-tag/WQ)—pLATE 52 vector, N-terminal His-tag protein expression, WELQut cleavage.
• aLICator™ LIC Cloning and Expression Set 1 (All-in-One/EK)—pLATE11, pLATE51 and pLATE31 vectors, choice of untagged, N- or C-terminal His-tag protein expression.
• aLICator™ LIC Cloning and Expression Set 2 (All-in-One/WQ)—pLATE11, pLATE52, and pLATE31 vectors, choice of untagged, N- or C-terminal His tag protein expression, WELQut cleavage.
For proteins with a known preference for either the N- or C-terminal 6xHis-tag position, using the appropriate N- or C-terminal kit is recommended. When the protein structure and features are not well known, it is recommended to clone into all three vectors and determine the most compatible vector for further research.
Related Products
aLICator LIC Cloning and Expression Kit 1 (untagged)
aLICator LIC Cloning and Expression Set 1 (All-in-One/EK)
aLICator LIC Cloning and Expression Kit 4 (N-terminal His-tag/WQ)
aLICator LIC Cloning and Expression Set 2 (All-in-One/WQ)
aLICator LIC Cloning and Expression Kit 3 (C-terminal His-tag)
规格
耐抗生素细菌
Ampicillin (AmpR)
克隆方法
LIC Cloning
适用于(应用)
PCR Cloning
反应次数
20
产品线
aLICator
产品类型
LIC Cloning and Expression Kit
促进剂
T7
蛋白标记
His Tag (6x)
载体
pLATE
产品规格
Kit
Thermo Scientific™
aLICator LIC Cloning and Expression Kit 3 (C-terminal His-tag)
货号: K1261
相关应用: PCR Cloning
技术支持客户服务
|
货号 |
单位规格 |
价格(CNY) |
库存状况 |
数量 |
|
K1261 |
20 reactions |
2,782.00 您的价格 登录 |
*** |
Thermo Scientific aLICator™ LIC Cloning and Expression System is designed for fast and efficient ligation independent cloning and tight regulation of gene expression in E. coli. The pLATE bacterial expression vectors are designed for high levels of target protein expression in concert with minimal background (uninduced) expression, which permits expression of proteins that are toxic to E. coli cells. To streamline and facilitate the process of insert cloning into the expression vector, the aLICator system uses directional LIC cloning technology, a rapid procedure that provides high-cloning efficiencies.
The tightly regulated expression and fast, efficient directional cloning makes the aLICator LIC Cloning and Expression System the best choice for routine and toxic gene cloning and expression in E. coli.
Highlights
• High efficiency LIC cloning
• Tight control of gene expression
• High yield expression
• Versatile—tagged or untaged protein expression with tag removal option
Applications
• Directional PCR product cloning
• Tightly regulated protein expression
• Expression of toxic genes
Principle
The aLICator LIC cloning system uses directional LIC cloning technology to streamline and facilitate cloning into an expression vector. LIC ensures high-cloning efficiencies of more than 95% and eliminates the need for ligation and restriction enzyme digestion steps.
The LIC method uses T4 DNA polymerase to create specific 14 to 21 nucleotide single-stranded overhangs on the pLATE vectors and DNA inserts. T4 DNA polymerase has two enzymatic activities: 5'→3' polymerase activity and 3'→5' exonuclease activity. The exonuclease activity removes nucleotides from the 3' ends of the DNA while the polymerase activity restores the chain using dNTPs and the complementary DNA strand as a template. In the LIC protocol, only dGTP is included in the reaction, causing the 3'→5'-exonuclease and 5'→3'-polymerase activities to equilibrate at the first occurrence of cytosine in the complementary strand. After annealing, the LIC vector and insert are transformed into competent E. coli cells without the use of T4 DNA ligase. Covalent bond formation at the vector-insert junctions occurs within the cell to yield circular plasmid.
Features
The system consists of four kits based on the pLATE series of bacterial expression vectors:
• aLICator™ LIC Cloning and Expression Kit 1 - pLATE11 vector, untagged protein expression.
• aLICator™ LIC Cloning and Expression Kit 2 (N-terminal His-tag/EK)—pLATE51 vector, N-terminal His-tag protein expression.
• aLICator™ LIC Cloning and Expression Kit 3 (C-terminal His-tag)—pLATE31 vector, C-terminal His-tag protein expression.
• aLICator™ LIC Cloning and Expression Kit 4 (N-terminal His-tag/WQ)—pLATE 52 vector, N-terminal His-tag protein expression, WELQut cleavage.
• aLICator™ LIC Cloning and Expression Set 1 (All-in-One/EK)—pLATE11, pLATE51 and pLATE31 vectors, choice of untagged, N- or C-terminal His-tag protein expression.
• aLICator™ LIC Cloning and Expression Set 2 (All-in-One/WQ)—pLATE11, pLATE52, and pLATE31 vectors, choice of untagged, N- or C-terminal His tag protein expression, WELQut cleavage.
For proteins with a known preference for either the N- or C-terminal 6xHis-tag position, using the appropriate N- or C-terminal kit is recommended. When the protein structure and features are not well known, it is recommended to clone into all three vectors and determine the most compatible vector for further research.
Related Products
aLICator LIC Cloning and Expression Set 1 (All-in-One/EK)
aLICator LIC Cloning and Expression Kit 4 (N-terminal His-tag/WQ)
aLICator LIC Cloning and Expression Set 2 (All-in-One/WQ)
aLICator LIC Cloning and Expression Kit 1 (untagged)
aLICator LIC Cloning and Expression Kit 2 (N-terminal His-tag/EK)
规格
耐抗生素细菌
Ampicillin (AmpR)
克隆方法
LIC Cloning
适用于(应用)
PCR Cloning
反应次数
20
产品线
aLICator
产品类型
LIC Cloning and Expression Kit
促进剂
T7
蛋白标记
His Tag (6x)
载体
pLATE
产品规格
Kit‘
Thermo Scientific™
aLICator LIC Cloning and Expression Kit 4 (N-terminal His-tag/WQ)
货号: K1281
相关应用: PCR Cloning
技术支持客户服务
Thermo Scientific aLICator™ LIC Cloning and Expression System is designed for fast and efficient ligation independent cloning and tight regulation of gene expression in E. coli. The pLATE bacterial expression vectors are designed for high levels of target protein expression in concert with minimal background (uninduced) expression, which permits expression of proteins that are toxic to E. coli cells. To streamline and facilitate the process of insert cloning into the expression vector, the aLICator system uses directional LIC cloning technology, a rapid procedure that provides high-cloning efficiencies.
The tightly regulated expression and fast, efficient directional cloning makes the aLICator LIC Cloning and Expression System the best choice for routine and toxic gene cloning and expression in E. coli.
Highlights
• High efficiency LIC cloning
• Tight control of gene expression
• High yield expression
• Versatile—tagged or untaged protein expression with tag removal option
Applications
• Directional PCR product cloning
• Tightly regulated protein expression
• Expression of toxic genes
Principle
The aLICator LIC cloning system uses directional LIC cloning technology to streamline and facilitate cloning into an expression vector. LIC ensures high-cloning efficiencies of more than 95% and eliminates the need for ligation and restriction enzyme digestion steps.
The LIC method uses T4 DNA polymerase to create specific 14 to 21 nucleotide single-stranded overhangs on the pLATE vectors and DNA inserts. T4 DNA polymerase has two enzymatic activities: 5'→3' polymerase activity and 3'→5' exonuclease activity. The exonuclease activity removes nucleotides from the 3' ends of the DNA while the polymerase activity restores the chain using dNTPs and the complementary DNA strand as a template. In the LIC protocol, only dGTP is included in the reaction, causing the 3'→5'-exonuclease and 5'→3'-polymerase activities to equilibrate at the first occurrence of cytosine in the complementary strand. After annealing, the LIC vector and insert are transformed into competent E. coli cells without the use of T4 DNA ligase. Covalent bond formation at the vector-insert junctions occurs within the cell to yield circular plasmid.
Features
The system consists of four kits based on the pLATE series of bacterial expression vectors:
• aLICator™ LIC Cloning and Expression Kit 1 - pLATE11 vector, untagged protein expression.
• aLICator™ LIC Cloning and Expression Kit 2 (N-terminal His-tag/EK)—pLATE51 vector, N-terminal His-tag protein expression.
• aLICator™ LIC Cloning and Expression Kit 3 (C-terminal His-tag)—pLATE31 vector, C-terminal His-tag protein expression.
• aLICator™ LIC Cloning and Expression Kit 4 (N-terminal His-tag/WQ)—pLATE 52 vector, N-terminal His-tag protein expression, WELQut cleavage.
• aLICator™ LIC Cloning and Expression Set 1 (All-in-One/EK)—pLATE11, pLATE51 and pLATE31 vectors, choice of untagged, N- or C-terminal His-tag protein expression.
• aLICator™ LIC Cloning and Expression Set 2 (All-in-One/WQ)—pLATE11, pLATE52, and pLATE31 vectors, choice of untagged, N- or C-terminal His tag protein expression, WELQut cleavage.
For proteins with a known preference for either the N- or C-terminal 6xHis-tag position, using the appropriate N- or C-terminal kit is recommended. When the protein structure and features are not well known, it is recommended to clone into all three vectors and determine the most compatible vector for further research.
Related Products
aLICator LIC Cloning and Expression Set 2 (All-in-One/WQ)
CloneJET PCR Cloning Kit
Fast DNA End Repair Kit
aLICator LIC Cloning and Expression Kit 1 (untagged)
规格
耐抗生素细菌
Ampicillin (AmpR)
克隆方法
LIC Cloning
适用于(应用)
PCR Cloning
反应次数
20
产品线
aLICator
产品类型
LIC Cloning and Expression Kit
促进剂
T7
蛋白标记
His Tag (6x)
载体
pLATE
产品规格
Kit
Thermo Scientific™
aLICator LIC Cloning and Expression Set 1 (All-in-One/EK)
货号: K1271
相关应用: PCR Cloning
技术支持客户服务
Thermo Scientific aLICator™ LIC Cloning and Expression System is designed for fast and efficient ligation independent cloning and tight regulation of gene expression in E. coli. The pLATE bacterial expression vectors are designed for high levels of target protein expression in concert with minimal background (uninduced) expression, which permits expression of proteins that are toxic to E. coli cells. To streamline and facilitate the process of insert cloning into the expression vector, the aLICator system uses directional LIC cloning technology, a rapid procedure that provides high-cloning efficiencies.
The tightly regulated expression and fast, efficient directional cloning makes the aLICator LIC Cloning and Expression System the best choice for routine and toxic gene cloning and expression in E. coli.
Highlights
• High efficiency LIC cloning
• Tight control of gene expression
• High yield expression
• Versatile—tagged or untaged protein expression with tag removal option
Applications
• Directional PCR product cloning
• Tightly regulated protein expression
• Expression of toxic genes
Principle
The aLICator LIC cloning system uses directional LIC cloning technology to streamline and facilitate cloning into an expression vector. LIC ensures high-cloning efficiencies of more than 95% and eliminates the need for ligation and restriction enzyme digestion steps.
The LIC method uses T4 DNA polymerase to create specific 14 to 21 nucleotide single-stranded overhangs on the pLATE vectors and DNA inserts. T4 DNA polymerase has two enzymatic activities: 5'→3' polymerase activity and 3'→5' exonuclease activity. The exonuclease activity removes nucleotides from the 3' ends of the DNA while the polymerase activity restores the chain using dNTPs and the complementary DNA strand as a template. In the LIC protocol, only dGTP is included in the reaction, causing the 3'→5'-exonuclease and 5'→3'-polymerase activities to equilibrate at the first occurrence of cytosine in the complementary strand. After annealing, the LIC vector and insert are transformed into competent E. coli cells without the use of T4 DNA ligase. Covalent bond formation at the vector-insert junctions occurs within the cell to yield circular plasmid.
Features
The system consists of four kits based on the pLATE series of bacterial expression vectors:
• aLICator™ LIC Cloning and Expression Kit 1 - pLATE11 vector, untagged protein expression.
• aLICator™ LIC Cloning and Expression Kit 2 (N-terminal His-tag/EK)—pLATE51 vector, N-terminal His-tag protein expression.
• aLICator™ LIC Cloning and Expression Kit 3 (C-terminal His-tag)—pLATE31 vector, C-terminal His-tag protein expression.
• aLICator™ LIC Cloning and Expression Kit 4 (N-terminal His-tag/WQ)—pLATE 52 vector, N-terminal His-tag protein expression, WELQut cleavage.
• aLICator™ LIC Cloning and Expression Set 1 (All-in-One/EK)—pLATE11, pLATE51 and pLATE31 vectors, choice of untagged, N- or C-terminal His-tag protein expression.
• aLICator™ LIC Cloning and Expression Set 2 (All-in-One/WQ)—pLATE11, pLATE52, and pLATE31 vectors, choice of untagged, N- or C-terminal His tag protein expression, WELQut cleavage.
For proteins with a known preference for either the N- or C-terminal 6xHis-tag position, using the appropriate N- or C-terminal kit is recommended. When the protein structure and features are not well known, it is recommended to clone into all three vectors and determine the most compatible vector for further research.
Related Products
aLICator LIC Cloning and Expression Kit 4 (N-terminal His-tag/WQ)
aLICator LIC Cloning and Expression Kit 3 (C-terminal His-tag)
aLICator LIC Cloning and Expression Set 2 (All-in-One/WQ)
aLICator LIC Cloning and Expression Kit 1 (untagged)
规格
耐抗生素细菌
Ampicillin (AmpR)
克隆方法
LIC Cloning
适用于(应用)
PCR Cloning
反应次数
30
产品线
aLICator
产品类型
LIC Cloning and Expression Kit
促进剂
T7
蛋白标记
His Tag (6x)
载体
pLATE
Thermo Scientific™
aLICator LIC Cloning and Expression Set 2 (All-in-One/WQ)
货号: K1291
相关应用: PCR Cloning
技术支持客户服务
|
货号 |
单位规格 |
价格(CNY) |
库存状况 |
数量 |
|
K1291 |
30 reactions |
3,014.00 您的价格 登录 |
*** |
Thermo Scientific aLICator™ LIC Cloning and Expression System is designed for fast and efficient ligation independent cloning and tight regulation of gene expression in E. coli. The pLATE bacterial expression vectors are designed for high levels of target protein expression in concert with minimal background (uninduced) expression, which permits expression of proteins that are toxic to E. coli cells. To streamline and facilitate the process of insert cloning into the expression vector, the aLICator system uses directional LIC cloning technology, a rapid procedure that provides high-cloning efficiencies.
The tightly regulated expression and fast, efficient directional cloning makes the aLICator LIC Cloning and Expression System the best choice for routine and toxic gene cloning and expression in E. coli.
Highlights
• High efficiency LIC cloning
• Tight control of gene expression
• High yield expression
• Versatile—tagged or untaged protein expression with tag removal option
Applications
• Directional PCR product cloning
• Tightly regulated protein expression
• Expression of toxic genes
Principle
The aLICator LIC cloning system uses directional LIC cloning technology to streamline and facilitate cloning into an expression vector. LIC ensures high-cloning efficiencies of more than 95% and eliminates the need for ligation and restriction enzyme digestion steps.
The LIC method uses T4 DNA polymerase to create specific 14 to 21 nucleotide single-stranded overhangs on the pLATE vectors and DNA inserts. T4 DNA polymerase has two enzymatic activities: 5'→3' polymerase activity and 3'→5' exonuclease activity. The exonuclease activity removes nucleotides from the 3' ends of the DNA while the polymerase activity restores the chain using dNTPs and the complementary DNA strand as a template. In the LIC protocol, only dGTP is included in the reaction, causing the 3'→5'-exonuclease and 5'→3'-polymerase activities to equilibrate at the first occurrence of cytosine in the complementary strand. After annealing, the LIC vector and insert are transformed into competent E. coli cells without the use of T4 DNA ligase. Covalent bond formation at the vector-insert junctions occurs within the cell to yield circular plasmid.
Features
The system consists of four kits based on the pLATE series of bacterial expression vectors:
• aLICator™ LIC Cloning and Expression Kit 1 - pLATE11 vector, untagged protein expression.
• aLICator™ LIC Cloning and Expression Kit 2 (N-terminal His-tag/EK)—pLATE51 vector, N-terminal His-tag protein expression.
• aLICator™ LIC Cloning and Expression Kit 3 (C-terminal His-tag)—pLATE31 vector, C-terminal His-tag protein expression.
• aLICator™ LIC Cloning and Expression Kit 4 (N-terminal His-tag/WQ)—pLATE 52 vector, N-terminal His-tag protein expression, WELQut cleavage.
• aLICator™ LIC Cloning and Expression Set 1 (All-in-One/EK)—pLATE11, pLATE51 and pLATE31 vectors, choice of untagged, N- or C-terminal His-tag protein expression.
• aLICator™ LIC Cloning and Expression Set 2 (All-in-One/WQ)—pLATE11, pLATE52, and pLATE31 vectors, choice of untagged, N- or C-terminal His tag protein expression, WELQut cleavage.
For proteins with a known preference for either the N- or C-terminal 6xHis-tag position, using the appropriate N- or C-terminal kit is recommended. When the protein structure and features are not well known, it is recommended to clone into all three vectors and determine the most compatible vector for further research.
Related Products
aLICator LIC Cloning and Expression Set 1 (All-in-One/EK)
aLICator LIC Cloning and Expression Kit 4 (N-terminal His-tag/WQ)
aLICator LIC Cloning and Expression Kit 3 (C-terminal His-tag)
aLICator LIC Cloning and Expression Kit 2 (N-terminal His-tag/EK)
aLICator LIC Cloning and Expression Kit 1 (untagged)
规格
耐抗生素细菌
Ampicillin (AmpR)
克隆方法
LIC Cloning
适用于(应用)
PCR Cloning
反应次数
30
产品线
aLICator
产品类型
aLICator LIC Cloning and Expression Set 2 (All-in-One/WQ)
促进剂
T7
蛋白标记
His Tag (6x)
载体
pLATE
Thermo Scientific™
WELQut 蛋白酶 (5 U/µL)
货号: EO0861
相关应用: Protein Purification & Isolation
技术支持客户服务
|
货号 |
单位规格 |
价格(CNY) |
库存状况 |
数量 |
|
EO0861 |
500 units |
1,448.00 您的价格 登录 |
*** |
Thermo Scientific WELQut 蛋白酶是金色葡萄球菌的高特异性重组丝氨酸蛋白酶。它可以识别并精确切割含有工程化识别序列 † W- E- L- Q↓X(Trp、Glu、Leu、Gln,X 可以是任何氨基酸)的重组蛋白质。蛋白酶在识别序列之外切割,不会使额外氨基酸与靶蛋白结合。
WELQut 蛋白酶在较宽的温度(4 至 30°C)和 pH 值(pH 值 6.5 至 9.0 )范围内均有活性,并且不需要特定的缓冲液。
此外,这种新的蛋白酶具有多种程序优势 - 它适用于柱上蛋白水解反应,可使用内置 His- 标签轻松地将其从反应混合物中去除。
产品优势
•在 WELQ 识别序列之外进行切割,不会使额外氨基酸与靶蛋白结合
•对同源识别位点具有高度特异性,即使在长时间孵育和使用过量蛋白酶之后也不会生成非特异性产物条带
•可使用内置 His- 标签轻松地将其从反应混合物中去除
•适用于柱上蛋白水解反应
应用
•从重组蛋白制剂中去除 N 端融合标签。
附注
†这一切割序列存在于表达载体 pLATE52中,包括在 Thermo Scientific 可提供的 Thermo Scientific aLICator LIC 克隆和表达试剂盒 4 中(N 末端 His 标签/WELQut)(#K1281)。
For Research Use Only. Not for use in diagnostic procedures.
规格
数量
500 U
类型
Protease
产品线
WELQut