Figure 1 : Method overview and primer design optimisation.

From: IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly

Figure 1

(a) Schematic of the universal IVA cloning protocol consisting of a single PCR reaction, producing homologous linear ends, followed by DpnI digestion and transformation, where amplified DNA is assembled in vivo by recombination. Primer design is shown for each type of basic modification: insertion, deletions, site-directed mutagenesis and sub-cloning. For insertions, the new sequence is best included in Fw and Rv primers, acting as the homologous region (magenta). For deletions, the overlap can be incorporated in any one primer, homologous to the other primer (orange) with primers straddling the undesired region (grey). Mutagenesis is similarly performed, inversely amplifying outside the undesired codon (ATG), with the replacement encoded in the forward primer (TGC). (b) Sub-cloning involves the amplification of both vector and insert in a single tube with homologous regions to directionally control assembly (blue and yellow).