CloneJET | Thermo Fisher Scientific

CloneJET PCR Cloning Kit

CloneJET PCR Cloning Kit is a positive selection system for efficient cloning of PCR products generated with any thermostable DNA polymerase.

Features

The CloneJET PCR Cloning Kit contains a novel, ready-to-use positive selection cloning vector pJET1.2pJET1.2/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 pJET1.2pJET1.2/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 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

For convenience in mapping and manipulation of the insert, the pJET1.2pJET1.2/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.

Note: 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.

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

pJET1.2pJET1.2/blunt Cloning Vector
T4 DNA Ligase
2X Reaction Buffer
DNA Blunting Enzyme
pJET1.2pJET1.2 Forward Sequencing Primer (5’-CGACTCACTATAGGGAGAGCGGC-3’)
pJET1.2pJET1.2 Reverse Sequencing Primer (5’-AAGAACATCGATTTTCCATGGCAG-3’)
Control PCR ProductpJET1.2
Water, nuclease-free
Detailed Protocol

Hazardous	No
Quality Control	The kit is functionally tested by cloning a control 3'-dA tailed PCR product. Typical yield of recombinant clones is >99%. The cloning vector is tested in a self-ligation experiment for the absence of cloning background. The pJET1.2 primers are tested for specific and efficient sequencing.
Storage Condition	-20ºC

Applications

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

What's included

pJET1.2pJET1.2/blunt Cloning Vector
T4 DNA Ligase
2X Reaction Buffer
DNA Blunting Enzyme
pJET1.2pJET1.2 Forward Sequencing Primer (5’-CGACTCACTATAGGGAGAGCGGC-3’)
pJET1.2pJET1.2 Reverse Sequencing Primer (5’-AAGAACATCGATTTTCCATGGCAG-3’)
Control PCR ProductpJET1.2
Water, nuclease-free
Detailed Protocol

Download sequences

Specifications

Hazardous	No
Quality Control	The kit is functionally tested by cloning a control 3'-dA tailed PCR product. Typical yield of recombinant clones is >99%. The cloning vector is tested in a self-ligation experiment for the absence of cloning background. The pJET1.2 primers are tested for specific and efficient sequencing.
Storage Condition	-20ºC

Supporting data

Procedure	Blunt PCR product	3'-dA talled PCR product
Blunting	--	5 min
Ligation	5 min	5 min
Total time	5 min	10 min

Table 1. CloneJET Cloning Procedure

pJET1.2/blunt vector map

Figure 1. pJET1.2/blunt vector map. pJET1.2/blunt cloning vector map. Click here for pJET1.2 sequence.

Efficiency of different blunt-end PCR cloning systems with positive selection

Figure 2. Cloning of blunt-end PCR product: efficiency of different blunt-end PCR cloning systems with positive selection. A 976 bp blunt-end PCR product, generated with Pfu DNA Polymerase, was directly ligated into different positive selection blunt-end PCR cloning vectors according to suppliers protocols. Ligation mixtures (2 µL) were used to transform E. coli DH10B cells. Transformation efficiency of competent cells was 1 x 107 transformants per µg supercoiled DNA.

Efficiency of different PCR cloning strategies for 3'-dA tailed PCR product

Figure 3. Cloning of 3’-dA tailed PCR product: efficiency of different PCR cloning strategies. A 976 bp 3’-dA tailed PCR product, generated with Taq DNA Polymerase, was ligated into pJET1.2/blunt and into different TA cloning vectors according to suppliers protocols. Ligation mixtures (2 µL) were used to transform E. coli DH10B cells. Transformation efficiency of competent cells was 1 x 107 transformants per µg supercoiled DNA.