Figure 4. One-tube multi-site modifications using IVA cloning.

(a) Schematic for multi-site modification whereby the position of a FLAG-tag (purple) is exchanged from the C- to the N-terminus of GluA2 (red) coding region in a CMV-based custom plasmid. The combination of deletion and insertion primers produces two amplification products after PCR. (b) The corresponding fragments (1 and 2) are visualised by agarose gel electrophoresis. (c) Schematic detailing multiple plasmid modification of GluA3-pRK5 vector in one tube. One set of primers a) deleted the N-terminal domain of GluA3 and b) inserted a FLAG-tag, while a second set of primers a) sub-cloned the TARP γ2 coding region (from a second vector) at the end of GluA3 and b) inserted a GSGSG linker to create a fusion construct. Together, these primers amplify three independent fragments, which are shown on an agarose gel (d). (d) Testing the number of multiple modifications that IVA cloning can perform simultaneously. Increasing number of XhoI restriction sites were created in the pRK5 plasmid using mutagenesis primers. Site of mutation is indicated by ▼. (f) PCR produced increasing numbers of bands corresponding to the number of modifications (1 to 1–5). (g) The number of colonies produced (yellow) and the percentage of correct clones (red) decreased with more modifications (n  =  3–5).