CloneJET PCR Cloning Kit

Flexible. Innovative. Fast.

The Thermo Scientific CloneJET PCR Cloning Kit is a versatile, advanced positive selection system for high-efficiency cloning of PCR products. Ligation into the positive selection vector takes only five minutes, yielding more than 99% recombinant clones. All common laboratory E.coli strains can be directly transformed with the ligation product. The CloneJET PCR Cloning Kit is also available as a combo kit provided with DH10B competent cells.

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Why use CloneJET PCR cloning kit?

Fast–clone PCR products in as little as 5 minutes
Versatile–use with phosphorylated or non-phosphorylated DNA fragments and with either blunt-end or sticky-end PCR products
Efficient–generate more than 99% of positive recombinant clones with no cloning background
Economical–eliminate the need for expensive blue/white screening
Compatible–transform directly into all common E. coli strains including TOP10 and XL1-Blue

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Principle of positive selection cloning

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The pJET1.2/blunt cloning vector carries 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. If the pJET1.2/blunt vector re-circularizes without an insert, it expresses the 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 pJET 1.2/blunt vector multiple cloning site contains two BgIII 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.

High efficiency

Blunt-end PCR products generated by proofreading DNA polymerases, such as Phusion DNA Polymerase, can be directly ligated into the vector in just 5 minutes. PCR products with 3'-A overhangs are blunted prior to ligation in 5 minutes using the proprietary thermostable DNA blunting enzyme included in the CloneJet PCR Cloning Kit. The cloning efficiency of both blunt- and sticky-end fragments was comparable between the CloneJET cloning kit and the ligase-based cloning kit (Vendor A) as shown in Figure 1. However, the cloning efficiency significantly decreased when using a topoisomerase-based cloning kit (Vendor B) to clone a blunt end DNA fragment.

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Figure 1. Cloning 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 positive control, the vendor-specific fragment was used. Ligation mixtures of 2 µl each were used to transform E. coli DH10B cells. Vendor-supplied competent cells were used for the corresponding topoisomerase-based reactions. Cloning efficiency was calculated based on percent of positive recombinants for the control.

Although the cloning efficiency of the CloneJET PCR cloning system is comparable to ligase-based PCR cloning kits, the transformation efficiency of recombinant clones derived using the CloneJET kit is 2- to 10-fold higher than clones derived from a ligase-based kit (Figure 2).

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Figure 2. 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.

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