Cold Fusion Cloning Kit

Seamless cloning of any DNA fragment into any vector at any location using the Cold Fusion range.

All Cold Fusion kits come complete with the enzyme mix and competent E. Coli cells for transformation of the fusion cloning reactions. Build your clones rapidly and easily.

How to clone using Cold Fusion

Experience the thrill of the “chill”—cloning in less than 20 minutes

The 5x Cold Fusion Master mix contains a proprietary blend of enzymes that prepare the ends of DNA fragments for sequence-directed alignment. Compatible DNA ends are then efficiently fused and produce vector clones with great accuracy.

PCR Amplification of Target DNA

To successfully clone any DNA fragment into a linearized vector, PCR primers must be designed to have at least 15 bases of homology with the end of the linearized vector. Thus, a primer will consist of a 15-base vector homology sequence at the 5′-end, and optional restriction site in the middle, and the gene-specific sequence at the 3′-end. The guidelines for primer designs are shown in the diagram below.

(Note: The restriction site in the middle of the primer can be the same or different one used to linearize the vector. You can also add any other sequence in the middle for frame adjustment or tag addition. For multiple DNA fragment joining, it is recommended that each PCR product shares at least 18 base pairs of homology.)

The PCR fragments can be generated by Taq DNA polymerase or other high fidelity DNA polymerase. The melting temperature (Tm) should be calculated based on the 3′ (gene-specific) end of the primer, NOT the entire primer.

Primers and primer dimers produced in PCR reactions are inhibitory to the Cold Fusion cloning reaction. If the PCR produces a single specific band (from an agarose gel), the PCR product can be purified by simply using a PCR purification kit. If the PCR produces multiple bands, the specific DNA band desired should be purified by a gel purification kit to remove non-specific DNA bands and avoid false-positive clones.

Cold Fusion cloning a single DNA fragment

Capable of cloning 3′, 5′ and blunt-end fragments efficiently

Cold Fusion cloning multiple DNA fragments

Capable of cloning multiple fragments in the order you choose

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