Cold Fusion Cloning Kit

Cold Fusion Cloning Kit

Key Features of the cloning technology

• Clone any insert, at any site within any vector
• Restriction enzyme, phosphatase and ligase free system
• Powerful technology to join multiple fragments at once
• Rapid one reaction for 5 minutes at room temperature, then fusion on ice for 10 minutes
• Introduce or correct mutations at will in cDNAs
• Amenable to high throughput cloning applications

Join the revolution in new cloning technologies

The Cold Fusion technology is a simple, rapid and highly efficient cloning kit. It allows you to directly clone any PCR product(s) to any linearized expression vector, at any site. The PCR fragments can be generated by Taq DNA polymerase or other high fidelity DNA polymerases, with primers that are designed to have at least 15 bases of homology at their linear ends to where you want the product to “fuse”. The linearized vector can be generated by PCR or restriction enzymes (single or double cut). One tube simple reaction format, 5 minute incubation at room temperature then 10 minutes on ice, your PCR product(s) rapidly and accurately fuse into the linearized vector in the desired orientation. The kit is so robust that multiple DNA fragments can be assembled simultaneously and cloned into one construct in a single step. The system is highly efficient, with more than 95% positive cloning rate.

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 areshown 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 othersequence in the middle for frame adjustment or tag addition. For multiple DNA fragment joining, it is recommended that each PCR productshares 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

Online Resources

Cold Fusion cloning kits Quantity Product Size Catalog # Price Cold Fusion Cloning Kit with Competent Cells 10 rxns MC010A-1 $215.00 Cold Fusion Cloning Kit with Competent Cells 20 rxns MC100A-1 $390.00 Cold Fusion Cloning Kit with Competent Cells 50 rxns MC101A-1 $900.00   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.

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