Thermo Fisher, K1232, CloneJET PCR Cloning Kit

40 Reactions 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 laboratoryE. colistrains can be directly transformed with the ligation product. FeaturesThe 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 hostE. colicell 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 forin vitroandin vivotranscription as well as sequencing of the insert. Highlights
•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 screeningApplications
• 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 vitroandin vivotranscription of cloned inserts from the T7 promoterIncludes
• pJET1. 2/blunt Cloning Vector
•T4 DNA Ligase
• 2X Reaction Buffer
• DNA Blunting Enzyme
• pJET1. 2Forward Sequencing Primer(5'-CGACTCACTATAGGGAGAGCGGC-3')
• pJET1. 2Reverse Sequencing Primer(5'-AAGAACATCGATTTTCCATGGCAG-3')
• Control PCR Product
•Water, nuclease-free
• Detailed ProtocolPrior to electroporation, always column-purify the ligation mixture using e. g. GeneJET PCR Purification Kit #K0701or chloroform to extract it. Electroporation is inhibited by the presence of proteins and salts in the mixture. Antibiotic Resistance Bacterial: Ampicillin (AmpR)
Cloning Method: Restriction Enzyme⁄ MCS, Blunt PCR
For Use With (Application): PCR Cloning
No. of Reactions: 40 Reactions
Product Line: CloneJET
Product Type: Cloning Kit
Quantity: 40 Reactions
Sample Type: PCR Products
Vector: pJET1.2/blunt
Format: Kit
Unit Size: Each