pENTR™/D-TOPO™ cloning kit utilizes a highly efficient 5-minute cloning strategy (“TOPO™ cloning”) to The end PCR product is directionally cloned and becomes the vector entering the Gateway™ system or MultiSite Gateway™ system. Blunt-end PCR products are directionally cloned with greater than 90% efficiency and require no ligases, post-PCR procedures, or restriction enzymes.

This kit contains everything needed to clone and select PCR-amplified genes of interest:

•Available for the Gateway™ System — Rapidly transfer cloned genes between multiple vector systems
• Fast and Easy — Go from PCR to Gateway™ in just 3 steps and as little as ∼5 minutes of hands-on time The process of getting started with cloning
• Efficient — achieves over 90% cloning with correct insertion in the right direction
• Proven — over the past decade Reliable performance

pENTR™ /D-TOPO™ Cloning Kit Overview

VECTOR:	pENTR™/D-TOPO™ vector	Directional cloning vector for entering the Gateway system
Cloning method	Directed TOPO™ Cloning	Ligate blunt-end proofreading polymerase-amplified PCR products to vectors via ∼5-minute topoisomerase I-based directional ligation
Competent cells	Two options	From kits containing high-efficiency or fast-growing competent cells Choose from

Easy access to the Gateway™ system
In order to enter the Gateway™ system, simply follow the PCR amplification gene and add the product directly to the provided topoisomerase charged pENTR™/D-TOPO™ vector, incubate for 5 minutes and transform the provided competent E. coli cells. The resulting attL containing Gateway™ entry clone is ready for efficient recombination and is available in Gateway™ destination vectors.

Optimized pENTR™/D-TOPO™ Vector
The PCR product insertion site of the pENTR™/D-TOPO™ vector (Figure 1) includes M13 and T7 on both sides Primer sequencing site and attL recombination site. This allows easy sequence verification of clones and recombination into various attR-containing Gateway™ destination vectors. Kanamycin resistance gene and pUC replication region sequences are used for selection and high-copy propagation in E. coli.

Simplified directional cloning
With directional TOPO™ cloning technology, there is no need for PCR purification, vector preparation, or other time-consuming DNA manipulation steps. Simply add the PCR reaction product directly to the provided topoisomerase charged vector, incubate for 5 minutes, transform and obtain up to 90% directional insertion clones. The four base overhangs on the vector pair with four base sequences were designed as forward primers for PCR reactions to provide directionality for the topoisomerase ligation reaction (Figure 2).

Performance of Gateway™ Recombinant Cloning Technology
Gateway™ Recombinant Cloning Technology avoids the limiting factors of restriction enzyme-mediated cloning, allowing you to easily create virtually All expression systems, 99% efficient and reversible Gateway™ recombination reaction. Because the same DNA sequence can be moved between different vectors, there is no need to use restriction enzymes, ligases, subcloning steps, screen countless colonies, or resequence, which will help you save time, money, and effort.

Leading cloning technology
In terms of cloning, TOPO™ cloning technology and Gateway™ recombinant cloning technology have been reliable partners for thousands of scientific researchers for ten years. Fast, easy-to-use, and efficient TOPO™ cloning and Gateway™ recombination enable rapid cloning and then transfer of genes between multiple Gateway™ expression vectors.