Thermo Scientific CloneJET PCR Cloning Kit, also available with DH10B cells (K123120), is an advanced positive selection system for high-efficiency cloning of PCR products generated with any thermostable DNA polymerase. Any other blunt or sticky-end DNA fragment can be cloned. It is ideal for phosphorylated or non-phosphorylated DNA fragments. Ligation into the included positive selection vector takes only 5 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 are blunted prior to ligation in 5 minutes with the thermostable DNA Blunting Enzyme provided with the kit. All common laboratory E. coli strains can be directly transformed with the ligation product.

Features

The CloneJET PCR Cloning Kit contains a 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. Recircularized 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.

For convenience in mapping and manipulation of the insert, the pJET1.2/blunt cloning vector multiple cloning site contains two BglII recognition sequences that flank the insertion site. In addition, the vector contains a T7 promoter for in vitro and in vivo transcription as well as sequencing of the insert.
Prior to electroporation, always column-purify the ligation mixture using e.g. GeneJET PCR Purification Kit #K0701 or chloroform to extract it. Electroporation is inhibited by the presence of proteins and salts in the mixture.

优点：

- Fast—PCR cloning in only 5 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

数据：

图1.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。 

图2. 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。 

图3.CloneJET Cloning Procedure

图4. Transformation efficiency of sticky- and blunt- end PCR products
A 976-bp PCR product generated with either Taq DNA Polymerase or Phusion DNA Polymerase was ligated into different cloning vectors according to the vendor’s recommended protocols. As a control, the vendor-specific fragment was used. Ligation mixtures of 2 µl each were used to transform E.coli DH10B cells. Transformation efficiency of competent cells was 1.2x10E7 transformants per µg supercoiled DNA.

组成成分：

▪ pJET1.2/blunt Cloning Vector
▪ T4 DNA Ligase
▪ 2X Reaction Buffer
▪ DNA Blunting Enzyme
▪ pJET1.2 Forward Sequencing Primer (5'-CGACTCACTATAGGGAGAGCGGC-3')
▪ pJET1.2 Reverse Sequencing Primer (5'-AAGAACATCGATTTTCCATGGCAG-3')
▪ Control PCR Product
▪ Water, nuclease-free
▪ Detailed Protocol

相关产品：

▪ DNA 片段标准品

▪ DNA ladder

▪ DNA 聚合酶

▪ PCR

▪ DNA纯化

▪ RNA纯化