Thermo Scientific™ CloneJET PCR Cloning Kit

Description

CloneJET PCR Cloning Kit 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.

The 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.

• 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

Recommended for:

Cloning of blunt-end or 3'-dA tailed PCR products up to 10kb; 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

Note:

Prior to electroporation always column-purify the ligation mixture using, e.g., GeneJET PCR Purification Kit, †K0701, or chloroform extract kit. Electroporation is inhibited by the presence of proteins and salts in the mixture.