AnyFusion® provides an extremely easy, efficient and rapid method for cloning. AnyFusion® contains an optimized blend of Genenmed®'s proprietary enzymes. AnyFusion® utilizes a ligase-independent homologous annealing system. The insert is prepared by PCR and directly coned into the linearized vector. For general cloning, the PCR-prepared insert does not need and further purification steps to remove the primers and/or dNTPs. The single-cut cloning vectors, even without further treatments, do not make a self-ligated background, and can be used as effectively as the double-cut vectors. The cloning efficiency and accuracy is almost 100%, ensuring that analysis of one or two colonies can complete cloning. AnyFusion® also clones multiple inserts. Therefore, AnyFusion® really establishes a new standard in cloning.
- Manufacturing Customized Premix with specific condition
- Be Made by One bath system
- Can be loaded on agarose gels without adding loading dye
- Reducing time
- Amplification up to 10Kb
1) AnyFusion® Protocol
1. Vector preparation
1) Cut the vector to linearize. Single or double cuts do not make any difference in cloning results. Complete digestion is crucial.
2) Heat-inactivate the restriction enzyme(s) used if it is heat-labile. If it is heat-resistant, use a PCR clean-up kit to remove the enzyme(s)
3) Use distilled water to adjust the vector concentration. 10~100ng of the linearized vector is used for AnyFusion® reaction.
2. Insert preparation
1) Perform PCR using Pfu polymerase (See Fig.3 for designing the primer). If the PCR template is a plasmid containing the same antibiotic resistance gene as the cloning vector, it can make a cloning background. To reduce it, pre-cutting the template vector before PCR is recommended.
2) If the PCR result shows only a specific product, no purification is needed. Otherwise, agarose gel purification is recommended. For cloning multiple inserts, purification using a PCR clean-up kit or through agarose gel electrophoresis is recommended. 10~100ng of the insert is used for AnyFusion® reaction.
[Designing PCR primers]
1. The 14 nucleotides homologous to the cloning vector is needed. The 10 nucleotides in length shows ~80% cloning efficiency.
2. To regenerate the restriction enzyme site, include the nucleotides in ( ) in Fig.3 to the primer sequence.
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