Thermo Scientific CloneJET PCR cloning kit can also be used with DH10B cells (K123120), is an advanced positive selection system that can efficiently clone PCR products generated by any thermostable DNA polymerase. Any other blunt or sticky end DNA fragment can also be cloned. It works well with phosphorylated or non-phosphorylated DNA fragments. Ligates to the included positive sorting vector in just 5 minutes and produces over 99% recombinant clones. The blunt-ended PCR product generated by the proofreading enzyme is directly inserted into the cloning vector.

PCR products generated by non-proofreading DNA polymerase or DNA polymerase mixtures are treated to blunt ends using the thermostable DNA blunting enzyme provided in the kit within 5 minutes before ligation. All common laboratory E. coli strains can be directly transformed using the ligation product.

Features

CloneJET PCR cloning kit contains a new ready-to-use positive selection cloning vector pJET1.2/blunt. The vector contains a lethal restriction enzyme gene that is destroyed after the DNA insert is inserted into the cloning site. Therefore, only bacterial cells containing the recombinant plasmid can form colonies. Recycled pJET1.2/blunt vector molecules without inserts express lethal restriction enzymes that kill host E. coli cells upon transformation. This positive selection greatly speeds up the colony screening process and saves the additional cost required for blue-white spot screening.

To facilitate mapping and processing of inserts, the multiple cloning site of the pJET1.2/blunt cloning vector contains two BglII recognition sequences flanking the insertion site. In addition, the vector contains the T7 promoter for in vitro and in vivo transcription as well as insert sequencing.

Product Advantages

•Fast—Complete PCR cloning in only 5 minutes
•Higher efficiency< /b>–>99% positive clones
•No cloning background—positive selection vector
•Multipurpose—either blunt-end or sticky-end cloning Ideal
•Economical—no need for expensive blue-white screening

Applications

•Clone up to 10 kb Terminal or '3-dA tail PCR products
•Clone restriction enzyme generated DNA fragments
•Clone DNA sequence
•From T7 Promoters for in vitro and in vivo transcription cloning inserts

includes

•pJET1.2/blunt cloning vector
• T4 DNA Ligase
• 2X Reaction Buffer
• DNA Blunt Endase
• pJET1.2 Forward sequencing primer (5'-CGACTCACTATAGGGAGAGCGGC-3')
• pJET1 .2 Reverse sequencing primer (5'-AAGAACATCGATTTTCCATGGCAG-3')
• Control PCR product
• nuclease-free water
•Detailed protocol< br>
Before electroporation, always use GeneJET PCR Purification Kit #K0701 to Column purification or extraction with chloroform. The presence of protein and salt in the mixture inhibits electroporation.

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CloneJET PCR cloning kit
Rapid DNA End Repair Kit