Invitrogen™ pENTR™/D-TOPO™ Cloning Kit, with One Shot™ TOP10 Chemically Competent E. coli

• Blunt-end PCR products clone directionally at greater than 90% efficiency, with no ligase, post-PCR procedures, or restriction enzymes required
• Rapidly shuttle cloned genes between multiple vector systems
• Reliable performance
• Directional cloning vector for entry to Gateway System
• For access to Gateway System, PCR amplify gene of interest and add product straight to provided topoisomerase charged pENTR/D-TOPO vector, incubate 5 minutes, and transform provided competent E. coli cells
• Resulting attL containing Gateway Entry clones are ready for efficient recombination with the choice of Gateway Destination vectors
• The pENTR/D-TOPO vector includes M13 and T7 primer sequencing sites and attL recombination sites flanking the PCR product insertion site, allowing clones to be easily sequence-verified and recombined into the choice of attR containing Gateway destination vectors
• Kanamycin resistance gene and a pUC origin are used for selection and high-copy propagation in E. coli
• Four base overhang on vector pairs with four base sequence designed into forward primer used in PCR reaction to provide directionality to the topoisomerase ligation reaction
• Circumvents the limitations of restriction mediated cloning, enabling to access virtually any expression system in a simple one hour, 99%-efficient and reversible, Gateway recombination reaction
• Move same sequence of DNA between different vectors without using restriction enzymes, ligase, subcloning steps, screening of countless colonies, or resequencing to save time, money, and effort

Cloning, Gateway Cloning