The pENTR™/D-TOPO™ Cloning Kits utilize a highly efficient, 5-minute cloning strategy (“TOPO™ Cloning”) to directionally clone a blunt-end PCR product into a vector for entry into the Gateway™ System or the MultiSite Gateway™ System. Blunt-end PCR products clone directionally at greater than 90% efficiency, with no ligase, post-PCR procedures, or restriction enzymes required.

The kit comes with everything necessary to clone and select your PCR amplified gene of interest:

• Gateway™ System Ready - Rapidly shuttle cloned genes between multiple vector systems
• Fast and Easy - Go from PCR to Gateway™ Entry clone in just 3 steps and as little as ∼5 minutes hands on time
• Efficient - Achieve over 90% clones with correct insert in the right direction
• Proven - Reliable performance for over a decade

pENTR™/D-TOPO™ Cloning Kits Overview

VECTOR	pENTR™/D-TOPO™ Vector	Directional cloning vector for entry to the Gateway System
CLONING METHOD	Directional TOPO™ Cloning	Topoisomerase I based ∼5 minute directional ligation of blunt-end proofreading polymerase-amplified PCR products to the vector
COMPETENT CELLS	Two Options	Choose from kits with either high efficiency, or fast growing competent cells

Simple Access to the Gateway™ System
For access to the Gateway™ System, just PCR amplify your gene of interest and add the product straight to the provided topoisomerase charged pENTR™/D-TOPO™ vector, incubate 5 minutes, and transform the provided competent E. coli cells. The resulting attL containing Gateway™ Entry clones are ready for efficient recombination with your choice of Gateway™ Destination vectors.

Optimized pENTR™/D-TOPO™ Vector
The pENTR™/D-TOPO™ vector (Figure 1) includes M13 and T7 primer sequencing sites and attL recombination sites flanking the PCR product insertion site. This allows clones to be easily sequence verified and recombined into your choice of attR containing Gateway™ destination vectors. A Kanamycin resistance gene and a pUC origin 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 clean up, vector preparation, or other time-intensive DNA manipulation steps. Just add your PCR reaction straight to the provided topoisomerase charged vector, incubate 5 minutes, transform, and obtain up to 90% directionally inserted clones. A four base overhang on the vector pairs with a four base sequence designed into the forward primer used in your PCR reaction to provide directionality to the topoisomerase ligation reaction (Figure 2).

The Power of Gateway™ Recombination Cloning Technology
Gateway™ recombination cloning technology circumvents the limitations of restriction mediated cloning, enabling you to access virtually any expression system in a simple one hour, 99%-efficient and reversible, Gateway™ recombination reaction. The ability to move the same sequence of DNA between different vectors without using restriction enzymes, ligase, subcloning steps, screening of countless colonies, or re-sequencing will help save you time, money, and effort.

Leading Cloning Technologies
When it comes to cloning, TOPO™ cloning technology and Gateway™ recombination cloning technology have been a reliable partner for thousands of scientists for over ten years. Fast, simple to use, and efficient, TOPO™ cloning and Gateway™ recombination allow for rapid cloning and subsequent transferring of genes between a wide assortment of Gateway™ expression vectors.

Kit Options
The pENTR™/D-TOPO™ Cloning Kit can be purchased with either TOP10 competent cells for standard cloning or Mach1™-T1R competent cells for fast growth.

For Research Use Only. Not intended for use in animal or human therapeutic or diagnostic use.