Restriction-free cloning

Key words Protein expression. Fusion-tags, Restriction-free cloning. Protein solubility, E. coli... 

In the present protocol, the restriction free (RF) cloning procedure (11,12) is described in detail. RF cloning can be used to introduce the same PCR product into multiple expression vectors harboring identical sequences at the sites of integration. All vectors listed in Fig. 1 are suitable for such parallel cloning. 

Unger, T, Jacobovitch Y, Dantes A, Bernheim R, Peleg Y. (2010) Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression. J Struct Biol 172 34 4... 

Examples of primer sequences for restriction free (RF) cloning of N-terminal Flis or His-TEV into pAcGP67, downstream to the signal peptide, are shown in Table 1. 

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