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Cold Fusion Cloning Kit with Competent Cells (international version)
/Product Detail : MC010B-1 Cold Fusion Cloning Kit with Competent Cells (international version)
Product name : Cold Fusion Cloning Kit with Competent Cells (international version)
Catalog number : MC010B-1
Quantity: 10 rxns
Supplier name : SBI
Data sheet : Ask more or other datasheet now !
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 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.
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